-aminobutyric 
 acid ( gaba ) is a major inhibitory neurotransmitter 
 in the mammalian central nervous system .
when gaba concentrations in the brain fall below a threshold level , 
 convulsions occur .
it has also been found 
 that alzheimer s disease , huntington s 
 chorea , parkinson s disease , and drug addiction are related to diminished gaba 
 levels in the brain .
gaba is produced by the -decarboxylation 
 of the excitatory neurotransmitter l - glutamic acid , catalyzed 
 by the pyridoxal 5-phosphate ( plp)-dependent enzyme glutamic 
 acid decarboxylase ( gad , scheme 1 ) .
gaba is 
 metabolized by -aminobutyric acid aminotransferase ( gaba - at ) , 
 also a plp - dependent enzyme , to succinic semialdehyde , which is further 
 oxidized to succinic acid by succinic semialdehyde dehydrogenase ( ssdh ) . 

degradation of gaba to succinic semialdehyde converts the plp coenzyme 
 to pyridoxamine 5-phosphate ( pmp ) ; -ketoglutarate is 
 used in a second step to return pmp to plp with concomitant formation 
 of l - glutamic acid .
therefore , the concentrations of the 
 inhibitory ( gaba ) and excitatory ( l - glu ) neurotransmitters 
 are regulated by the appropriate balance between gad and gaba - at . when gaba is injected directly 
 into the brain of a convulsing animal , 
 the seizures are terminated .
however , 
 because gaba does not cross the blood  brain barrier , peripheral 
 gaba administration is not an effective approach for the treatment 
 of epilepsy .
an alternative approach to increase gaba levels in the 
 brain is by inhibition of gaba - at , which blocks the degradation of 
 gaba .
a mechanism - based inactivator , 
 an unreactive compound that initially acts as a substrate for the 
 target enzyme and is converted to a species that causes inactivation 
 of that enzyme , generally by covalent attachment , could increase gaba 
 levels for an extended period of time .
the design of these types of inhibitors requires insight into the 
 mechanism of the target enzyme so that the catalytic chemistry can 
 be utilized in the activation of the inactivator ; the mechanism of 
 the first half of the  ping - pong  reaction catalyzed 
 by gaba - at is shown in scheme 2 .
incubation of gaba with gaba - at leads to the conversion 
 of a lysine - plp 
 aldimine ( 1 ) to a gaba - plp aldimine ( 2 ) . 

abstraction of the -proton by lys329 and tautomerization leads 
 to a pmp aldimine ( 3 ) ; enzyme - catalyzed hydrolysis of 
 this intermediate leads to succinic semialdehyde and pmp .
4-amino-5-hexenoic acid ( 4 , -vinyl - gaba ; vigabatrin ) 
 is a mechanism - based inactivator of gaba - at that is an fda - approved drug ( sabril ) to treat infantile spasms 
 and refractory partial seizures .
however , because of a serious side 
 effect , namely , retinal damage and blindness , a drug with more potency 
 and less toxicity was desired .
mechanistic studies performed by nanavati 
 and silverman ( scheme 3 ) demonstrated that vigabatrin inactivates gaba - at via two 
 mechanisms , a michael addition reaction and an enamine reaction , in 
 a ratio of 70:30 . in the michael addition pathway ( pathway a ) , vigabatrin 
 acts like substrate gaba and undergoes aldimine formation with plp , 
 deprotonation , and tautomerization to a michael acceptor ( 5 ) , which undergoes michael addition by lys329 , the lysine residue 
 to which plp is bound in the native enzyme , giving , after protonation , covalent adduct 6 . enzyme - catalyzed 
 hydrolysis of 5 gives ,-unsaturated ketone 7 , which may be a possible cause for side effects of the drug . 
 in the enamine pathway ( pathway b ) , an enamine intermediate ( 8) , formed by tautomerization through the alkene , attacks 
 the enzyme - bound plp to inactivate gaba - at ( 9 ) , also 
 by formation of a covalent adduct .
hydrolysis of 8 gives 
 a weak electrophile , which is unlikely to proceed through further 
 reactions in the active site . to avoid the potentially 
 toxic intermediate ( 7 ) , computer 
 modeling based on the crystal structures of gaba - at and the vigabatrin 
plp complex in gaba - at was carried out to determine how to prevent 
 the michael addition pathway .
the model predicted that , in the energy - minimized 
 structure , the alkene double bond pointed away from the nucleophilic 
 lys329 ; for michael addition to occur , that bond had to rotate , allowing 
 the alkene to be aligned with the side chain amine of lys329 .
a conformationally 
 rigid vigabatrin analogue was prepared ( 10 ) to prevent 
 michael addition , and it was found that it inactivated gaba - at exclusively 
 by the enamine pathway ; however , it was 
 a weak inactivator .
the second approach to prevent formation of reactive 
 product 7 was to synthesize a vigabatrin analogue that 
 would give rapid michael addition so that the equivalent of intermediate 5 would not be sufficiently stable to allow the enzyme - catalyzed 
 hydrolysis leading to the equivalent of 7 .
that approach required the synthesis of a compound 
 with the alkene pointing toward the side chain amine of lys329 ( 11 ) .
the obtained compound inactivated gaba - at , but only in 
 the absence of the electrophilic trapping agent 2-mercaptoethanol . 

therefore , an undesirable reactive intermediate ( the corresponding 
 ketoacid of 11 ) was formed prior to inactivation . to 
 accelerate the inactivation rate , two electron - withdrawing fluorine 
 atoms were attached to the alkene of 11 , and the obtained 
 compound ( 12 ) inactivated gaba - at , even in the presence 
 of 2-mercaptoethanol .
further studies with 12 ( now called 
 cpp-115 ) showed that it was at least 187 times more potent an inactivator 
 of gaba - at than vigabatrin , was not active in the cerep panel of 111 
 pharmacological targets ( in vitro assays , carried out commercially 
 by cerep , on cpp-115 that cover a broad range of targets , including 
 receptors , ion channels , enzymes , transporters , and second messengers ) , 
 does not bind to three human gaba transporters gat1 , gat2 , and gat3 , 
 or to gabaa , gabab , or gabac receptors , 
 does not bind to the herg channel , does not inhibit or induce cytochrome 
 p450s , is not metabolized by hepatocytes , is rapidly and completely 
 orally absorbed and eliminated , has no adverse effect on respiration , 
 produces no mutations or chromosomal aberrations , and has a considerably 
 lower retinal toxicity liability .
it 
 also is 300600 times more potent than vigabatrin in reducing 
 cocaine - induced synaptic dopamine levels and in blocking expression 
 of cocaine - induced conditioned place preference in freely moving rats . 

recently , it was shown to have no adverse effects in a phase i clinical 
 trial .
because of the clinical importance of this new drug candidate , 
 we studied its inactivation mechanism to determine if it indeed favors 
 the michael addition pathway for which it was designed .
we have determined 
 that cpp-115 inactivates gaba - at via an unexpected inactivation mechanism , 
 namely , enzyme - catalyzed conversion of the difluoromethenyl group 
 to a carboxylate , which causes a conformational change in the enzyme , 
 to give a tightly bound complex .
this inactivation mechanism presents 
 a novel approach to inactivate gaba - at and other related enzyme targets 
 using similar catalytic mechanisms .
the turnover number for inhibition was determined by incubating 
 gaba - at with various equivalents of cpp-115 .
figure 1 shows the remaining enzyme activity versus time for 04 
 equiv of cpp-115 . to determine the turnover number , the data points 
 at 24 h were replotted as a pseudo - first - order reaction using the 
 remaining enzyme activity versus equivalents .
however , as shown 
 in the replot , nonpseudo - first - order inactivation kinetics was observed , 
 possibly because of product inhibition . ( top ) 

( bottom ) determination of the turnover number for the 
 inactivation of gaba - at by cpp-115 .
the data points at 24 h are replotted 
 as enzyme activity vs equiv of cpp-115 . to test
if the mechanism involves irreversible and/or 
 reversible inhibition , the time - dependent reactivation of gaba - at 
 was studied .
first , the enzyme was treated with an excess of inactivator ; 
 then , the mixture was dialyzed against bulk buffer .
return of enzyme activity 
 was observed , which displayed both time- and concentration - dependence 
 ( see supporting information , figures s1 
 and s2 ) .
the data also show that the amount of activity of the inhibited 
 gaba - at that returned was dependent on the amount of cpp-115 used 
 in the incubation and the incubation time . in figure s1
( supporting information ) , up to 90% and 60% of 
 enzyme activity was recovered when gaba - at was inactivated with 40 
 and 80 equiv of cpp-115 , respectively , for 3 h. however , when gaba - at 
 was incubated for 24 h , even with 10 equiv of cpp-115 , only 50% of 
 the activity returned ( figure s2 , supporting information ) . in the presence of 1000 equiv ( approximately 1 mm ) of cpp-115 
 in the incubation mixture ,
the activity of the enzyme returned to 
 about 8% ; with 2000 equiv of cpp-115 , the activity return was only 
 1% , even after 50 h of dialysis .
upon incubation of gaba - at with cpp-115 , fluoride 
 ions were continually released , even after the enzyme s activity 
 had fallen to almost zero ( figure s3 , supporting 
 information ) .
after normalization of the values with the controls , 
 it was found that 66  3 equiv of fluoride ions were released 
 slowly over a period of 27 h. however , when the same experiment was 
 repeated with plp supplemented in the mixture , there were 127  
 5 equiv of fluoride ions released over time . because -ketoglutarate 
 is essential to regenerate plp from pmp ,
if -ketoglutarate 
 is omitted , the amount of fluoride ions released per single turnover 
 of inhibition can be determined .
the continual release of fluoride 
 ions was not observed when there was no -ketoglutarate present , 
 and only 4.3  0.5 equiv of fluoride ions were detected per enzyme 
 dimer ( figure s4 , supporting information ) .
however , when -ketoglutarate was added to the inactive 
 enzyme , even without supplementation with plp , the fluoride ion concentration 
 began to increase .
a control experiment with no enzyme in the incubation 
 mixture was performed , which confirmed that no fluoride ions were 
 released from cpp-115 in the absence of the enzyme , even if plp is 
 added . to determine the fate of the coenzyme by inactivation 
 with cpp-115
the maximum recovered activity was 71.4% of the original , and the 
 activity recovery plateaued after 5 h of the reactivation process 
 ( table 1 ) , indicating that 29.6% of the enzyme 
 remained inactive .
[ h]plp - gaba - at was inactivated with cpp-115 , 
 and the 
 released radioactive compounds were analyzed using hplc ( figure s5 , supporting information ) .
the experiment 
 was performed with two controls : cpp-115 was omitted in a negative 
 control ( all radioactivity should be labeled plp ) , and gaba replaced 
 cpp-115 with -ketoglutarate omitted in a positive control ( all 
 radioactivity should be pmp ) . the negative control released most of 
 its radioactivity as plp ( but none as pmp ) , whereas a gaba - incubated 
 control released both pmp and plp ( figure s6 , supporting information ) .
given that the positive control with gaba 
 should only produce pmp from active enzyme , the plp present in this 
 sample represents that released from the inactive portion of gaba - at . 

after the background radioactivity from the control experiments was 
 subtracted from the cpp-115 experiment , cpp-115-inactivated [ h]plp - reconstituted gaba aminotransferase was found to release 
 100% of its cofactor as [ h]pmp ( figure 
 s7 , supporting information ) .
the small radioactive peak at 
 33 min was confirmed to be degraded plp present in all of the samples .
after inactivation of gaba - at by cpp-115 
 followed by denaturation and filtration , three metabolites were identified 
 from the sample solution that were not present in the control samples : 
 [ m / z ] 127.0389 , 171.0291 , and 401.0745 
 ( a , b , and c in figure s8 , supporting information , respectively ) , corresponding to 22 , 21 , and 20 , respectively , in scheme 5 ( see discussion ) .
fragmentation 
 data for m / z 127.0389 ( supporting information figure s9 ) confirmed the 
 structure of 3-oxo-1-cyclopentanecarboxylic acid ( 22 ) . 

ms / ms for m / z 171.0291 and 401.0745 
 ( supporting information figures s10 and s11 , respectively ) confirmed the structures of 21 and 20 . since covalent 
 modification of gaba - at was not detected using middle down proteomics 
 ( see figures s12s14 in supporting information ) , we analyzed the intact gaba - at protein to see if any detectable 
 mass shifts were present .
the intact mass data showed multiple peaks , 
 indicating that the gaba - at purified from pig brain was a mixture 
 of gaba - at species with different n - termini .
three samples were tested 
 using lc / ms / ms : free enzyme ( negative control ) , vigabatrin - inactivated 
 gaba - at ( positive control ) , and cpp-115-inactivated gaba - at ( figure s15 , supporting information ) .
vigabatrin - inactivated 
 gaba - at showed an added mass of 122 da from the mass of the native 
 enzyme , which matches the covalent adduct proposed previously .
however , cpp-115-inactivated gaba - at showed 
 no significant peaks corresponding to any added mass . to stabilize 
 any potential imine adducts throughout the lc / ms / ms process , the samples 
 were reduced with sodium borohydride as described previously for crystallography 
 studies .
reduction resulted in stabilization of the plp cofactor on 
 the enzyme , with an added mass of 236 from the original peak ( m ) . 
 as expected , the peaks of vigabatrin - inactivated gaba - at ( figure s16 , supporting information ) were no different 
 from those in figure s15 since the vigabatrin 
 covalent adduct is stable . interestingly , 
 for cpp-115-inactivated
gaba - at , reduction had no effect on the resulting 
 data and showed no added mass on the protein . to understand how 
 time - dependent inactivation of gaba - at by cpp-115 
 could occur without covalent modification of the protein or cofactor , 
 cpp-115-inactivated and dialyzed
the crystal 
 structures of native gaba - at from pig brain and inactivated enzyme 
 were obtained at 1.63  and 2.19  resolution , respectively . 

the crystal structure for the native pig brain enzyme was very similar 
 to that reported from pig liver by storici et al .
the crystal structures of the native enzyme and the inactivated 
 enzyme were compared to analyze the difference in overall structure 
 ( figure 2 ) and in the active site ( figure 3 ) .
the active site of the inactivated 
 gaba - at was investigated to understand the ligand 
enzyme interactions 
 ( figure 4 ) ; the omit map supports the ligand 
 interpretation ( see supporting information , figure s17 ) .
ribbon diagram of the superimposed native gaba - at ( yellow ) 
 and 
 gaba - at ( cyan ) bound to cpp-115 .
superimposition of the crystal structures of native gaba - at ( pink ) 
 and cpp-115-inactivated gaba - at ( green ) .
stereoview of gaba bound by the cpp-115 adduct . the 2fo  fc electron - density 
 map is shown as light gray mesh at 1.1 level around the cpp-115 
 adduct .
at 
 the start of this investigation , we considered a variety of 
 likely mechanisms for the inactivation of gaba - at by cpp-115 ( schemes 48 ) and then considered experiments to differentiate them .
all of these 
 inactivation mechanisms are initiated by the same steps shown in scheme 2 for substrate turnover or the two inactivation 
 mechanisms for vigabatrin shown in scheme 3 . in mechanism 1 ( scheme 4 ) , following -proton 
 abstraction of 13 , tautomerization leads to an ,-unsaturated 
 intermediate ( 14 ) , which is attacked by the active site 
 lysine residue or another base to covalently modify the enzyme .
no 
 fluorine is released , the enzyme is inactivated , and hydrolysis of 15 gives 16 with release of pmp as the cofactor .
if intermediate 15 in scheme 4 ( where x is either the active 
 site lysine residue or oh from attack 
 by water ) eliminates one fluoride ion ( scheme 5 ) , the inactivator can form another reactive ,-unsaturated 
 imine ( 17 ) , which can be attacked by a water molecule 
 to release the other fluoride ion , forming 18 .
depending 
 on what x is , this may result in inactivation or turnover . if x is 
 the active - site lysine residue ( 15a , pathway a , scheme 5 ) , the intermediate is a stabilized amide ; hydrolysis 
 generates covalent adduct 19 with the release of pmp
. 
 however , if x is a hydroxyl group ( 15b , pathway b , scheme 5 ) formed by water molecule attack 
 on reactive intermediate 14 ( scheme 4 ) , stable dicarboxylate intermediate 20 will 
 form ; hydrolysis gives metabolite 21 and pmp , and subsequent 
 decarboxylation of 21 gives 22 .
the third potential mechanism 
 involves allylic tautomerization 
 of external aldimine 13 to form 23 ( scheme 6 ) .
because 23 also 
 is a reactive electrophile , it may undergo michael addition to form 
 adduct 24 , which could be hydrolyzed to give 25 and release pmp . in the fourth potential 
 mechanism ( scheme 7 ) ,
pathway a generates an enamine ( 26 ) , similar to that 
 formed during vigabatrin inactivation ; this species might undergo 
 enamine attack on the lys329-bound plp to form covalent adduct 27 , which could hydrolyze to 28 . in pathway b , 

one fluoride ion is released by nucleophilic substitution ( 29 ) before undergoing transimination to form enamine adduct 30 , which might undergo hydrolysis to 31 .
pathway c from 30 involves elimination of a second fluoride ion ( 32 ) followed by hydrolysis to give 33 .
the final potential mechanism involves fluoride 
 ion elimination 
 from enamine 26 to generate reactive michael acceptor 34 ( scheme 8) . 

attack by an active site nucleophile ( either lys329 or 
 water ; pathway b ) gives either covalent adduct 36 or 
 noncovalent adduct 37 , respectively .
elimination of another 
 fluoride ion forms 38 or 40 , each of which 
 can be subsequently hydrolyzed to covalent adduct 39 or 
 metabolite 41 ; further hydrolysis of 39 would 
 also give 41 . in either case , this mechanism releases 
 the cofactor as plp .
all of these possible mechanisms can be differentiated 
 by determination of the number of fluoride ions released , the fate 
 of the cofactor , and the final metabolites or adducts formed ; these 
 possibilities are summarized in table 2 .
the partition ratio , the number of inactivator turnovers 
 per inactivation 
 event , was investigated by determination of the number of molecules 
 of cpp-115 that were required to cause complete enzyme inactivation . 

time - dependent inhibition was observed for 14 equiv of cpp-115 , 
 although pseudo - first - order kinetics was not observed ( figure 1 , top ) .
a replot of these data with extrapolation 
 of the first few data points gave a turnover number of 1.3  
 0.3 per active site ( figure 1 , bottom ) .
the 
 nonpseudo - first - order inactivation kinetics displayed indicated possible 
 product inhibition and suggested possibly two concurrent mechanisms 
 occurring at different rates , maybe one reversible and one irreversible . to test whether there was a reversible component to the inactivation
, 
 the titration experiment was repeated , first with 080 equiv 
 of cpp-115 ( figure s1 , supporting information ) , then 02000 equiv of cpp-115 ( figure 
 s2 , supporting information ) , followed by dialysis .
aliquots 
 removed during dialysis exhibited time - dependent reactivation of gaba - at 
 over an extended period of time ; with increasing equiv of inactivator , 
 less activity returned with maximal return by 24 h ( maximal return 
 of activity occurred sooner with less inactivator ) .
it required about 
 2000 equiv of cpp-115 before the enzyme reactivation was diminished 
 to an insignificant level , supporting a reversible and irreversible 
 component to the inactivation . during inactivation of gaba - at 
 by 2 mm cpp-115
, it was found that 
 66  3 equiv of fluoride ions were released over a 26 h time 
 period ; however , enzyme activity was lost within minutes ( figure s3 , supporting information ) .
this result 
 further supports the notion of two different mechanisms being involved , 
 one reversible and one irreversible , but it does not resolve the question 
 of how many fluoride ions are released per molecule .
a single - turnover 
 experiment was carried out to determine the number of fluoride ions 
 per molecule released with each turnover .
this experiment is possible 
 because gaba - at converts substrate to product with concomitant conversion 
 of its plp to pmp .
the reaction cycle can not restart until -ketoglutarate 
 binds and converts the pmp back to plp with concomitant formation 
 of l - glutamic acid . by leaving out the -ketoglutarate , 

4.3  0.5 equiv of fluoride ions was detected ( figure s4 , supporting information ) ; because gaba - at is a homodimer , 
 two molecules of cpp-115 can be turned over per enzyme dimer .
therefore , 
 4 equiv of fluoride ion indicates that both fluoride ions in cpp-115 
 are lost per enzyme turnover , and mechanisms 1 , 3 , and 4a , which release 
 no fluoride ions during inactivation , and mechanism 4b , which releases 
 one fluoride ion , can be excluded ( table 2 ) . the cofactor structure after inactivation by cpp-115 was determined 
 using gaba - at containing [ h]plp ( figure 
 s5 , supporting information ) . with the aid of two control experiments 
 ( figure s6 , supporting information )
, it 
 was found that all of the cofactor was converted to pmp after inactivation 
 ( figure s7 , supporting information ) .
therefore , 
 mechanism 4a , which does not release any cofactor and mechanisms 4b , 
 4c , 5a , and 5b , which require the release of plp as a cofactor , can 
 be excluded ( table 2 ) . of the mechanisms 
 considered ,
high - resolution lc / ms identified parent 
 ions with m / z 401.0745 , 171.0291 , 
 and 127.0389 ( figure s8 , supporting information ) , which corresponds to 20 , 21 , and 22 , respectively .
ms / ms fragmentation produced daughter ions 
 ( figures s9s11 , supporting information ) consistent with these three products and with mechanism 2b .
this 
 mechanism appears to be responsible for the reversible component of 
 the overall inactivation mechanism ; mechanism 2a would be expected 
 to account for the irreversible component , so it was imperative to 
 demonstrate covalent attachment to the enzyme ( 19 , scheme 5 ) for substantiation .
mass spectrometry of 
 the intact mass of cpp-115-inactivated gaba - at 
 was carried out using lc / ms / ms on native enzyme as the negative control 
 and vigabatrin - inactivated gaba - at as the positive control ( figure s15 , supporting information ) .
compared 
 with native enzyme , vigabatrin - inactivated gaba - at had an added m / z peak of 122 da , which corresponds to 
 the expected added mass of the covalent adduct previously proposed .
cpp-115-inactivated gaba - at showed no significant 
 peaks corresponding to added mass . in case the potential covalent 
 adduct with cpp-115 was an imine , the inactivated enzyme was reduced 
 with sodium borohydride prior to lc / ms / ms ( figure 
 s16 , supporting information ) .
again , no added mass was detected 
 with the cpp-115 inactivated enzyme , but the vigabatrin - inactivated 
 enzyme had the expected added mass .
these results were corroborated 
 by results from peptide proteomics ( supporting 
 information , figures s13 and s14 ) .
the 
 solution to this dilemma came from the x - ray crystal structure 
 of cpp-115-inactivated pig brain gaba - at , which revealed that the 
 inactivator was tightly bound to the protein noncovalently as 20 ( figures 3 and 4 ) .
the inactivated species binds tightly ( stable to dialysis ) 
 because of its covalent attachment to the cofactor and by two strong 
 electrostatic interactions between the guanidinium groups of arg192 
 and arg445 and each of the two carboxylate groups of 20 .
this unexpected phenomenon is the first time that arg445 has been 
 observed interacting directly with a ligand and to be involved in 
 the inactivation of gaba - at . on the basis of crystallography of gaba 
 analogues
, it is believed that gaba binds in the active site by schiff 
 base formation with plp and an electrostatic interaction between the 
 carboxylate of gaba and the guanidinium group of arg192 .
arg445 is 
 sequestered from the active site by an electrostatic interaction with 
 glu270 .
it has been proposed that this 
 second salt bridge only disassociates during the second half of catalysis , 
 the regeneration of plp from pmp , to aid in the binding of the second 
 carboxylate of -ketoglutarate .
a network of hydrogen bonds 
 in the active site of gaba - at connects glu270 and arg445 to o3 
 of the cofactor through the side chain amide of gln301 .
proposed that this network may be affected when the cofactor is 
 in its pmp form , which weakens the interaction between glu270 and 
 arg445 . to verify these hypotheses ,
many 
 research groups have attempted to cocrystallize gaba - at with ligands 
 such as -kg or glutamate that may directly show the involvement 
 of this second arginine , but they have not yet been successful .
our crystal structure of gaba - at inactivated by cpp-115 ( figure 3 ) clearly shows for the first time that the salt 
 bridge between arg445 and glu270 has been broken , and glu270 is rotated 
 away from its original position to accommodate a second guanidinium 
carboxylate 
 electrostatic interaction between arg445 and the newly formed carboxylate 
 group on 20 from enzyme - catalyzed hydrolysis of the difluoromethenyl 
 group .
the two guanidinium  carboxylate interactions , the hydrophobic 
 interactions of the pyridine ring , and the interactions of the phosphate 
 group contribute to the stabilization of the metabolite for tight 
 binding . therefore , cpp-115 appears to be , to the best of our knowledge , 
 the first example of a mechanism - based inactivator that leads to a 
 conformational change , forming a tightly bound inhibitor 
apparently , this conformational 
 change occurs only about once with 2000 molecules of inactivator ; 
 the remaining molecules of 2022 are 
 slowly released as products . the ,-unsaturated 
 intermediate ( 14 , 
 scheme 4 )
generated during turnover is highly 
 electrophilic , which should have been attacked by the nucleophilic 
 lys329 residue and form a covalent adduct , as it does with vigabatrin . 

molecular modeling studies of the cpp-115plp aldimine were 
 performed to analyze the effect of tautomerization during the inactivation 
 of gaba - at by cpp-115 ( figure 5 ) .
this model 
 demonstrates that the pmp - imine form ( orange ) of the aldimine generated 
 during enzyme - catalyzed tautomerization shifts the difluoromethenyl 
 group ( fluorines are in green ) away from lys329 , thereby moving it 
 too far for michael addition by this lysine residue . because of the 
 lack of 1,5 steric interactions ( both carbons opposite the carboxylate 
 group are sp - hybridized and thus planar ) , the difluoromethenyl 
 group is able to release strain by moving closer to the carboxylate . 
 as gaba - at normally catalyzes hydrolysis of the imine formed by tautomerization 
 of gaba ,
the catalytic machinery is in place for water attack of the 
 michael acceptor instead .
this is consistent with the obtained crystal 
 structure and explains why the inactivation mechanism did not follow 
 that of vigabatrin and form a covalent adduct .
molecular docking studies 
 of cpp-115-plp aldimine in plp - form ( cyan ) 
 vs cpp-115-pmp - form ( orange ) in the active site of gaba - at . on the basis of the evidence from 
 fluoride release , cofactor release , 

metabolites formed , proteomics , and x - ray crystallography , the most 
 consistent mechanism is shown in scheme 9 .
although cpp-115 was rationally designed 
 to inactivate gaba - at 
 via a covalent michael addition mechanism , the results described here 
 indicate that it inactivates gaba - at by mechanism - based formation 
 of a metabolite that induces a conformational change and forms a tightly 
 bound complex with the enzyme ( scheme 9 ) .
the 
 requirement of about 2000 equiv of cpp-115 for complete inactivation 
 of gaba - at reflects the number of molecules of product that are released 
 prior to interception by the enzyme in the correct state and conformation 
 that lead to inactivation .
the crystal structure of cpp-115-inactivated gaba - at revealed , 
 for the first time , that the glu270-arg445 salt bridge in the active 
 site can be disrupted , leading to the formation of a new binding pocket 
 for the inactivator .
our molecular modeling studies indicate a movement 
 of the difluoromethenyl group upon tautomerization , which shifts that 
 group too far from lys329 for nucleophilic attack .
this leads to enzyme - catalyzed 
 hydrolysis of the pmp - aldimine of cpp-115 and either release of products 
 ( 2000 times ) or inducement of a conformational change to give a tightly 
 bound pmp complex stabilized by the exposure of arg445 and formation 
 of an electrostatic interaction with the newly formed carboxylate 
 of the inactivator .
once metabolite 20 is tightly bound 
 to the enzyme , it is released from the active site very slowly , which 
 accounts for the extended period of time that fluoride ions are released 
 relative to the rate of inhibition of the enzyme . with our results 
 revealing the possibility of additional binding with arg445 ,
future 
 inactivators may be designed so that the newly formed carboxylate 
 group more closely mimics the second carboxylate group of -ketoglutarate 
 to encourage less turnover prior to conformational change - induced 
 exposure of arg445 .
gaba - at assays were recorded on 
 a synergy h1 hybrid multimode microplate reader ( biotek , usa ) with 
 transparent 96-well plates ( greiner bio - one , monroe , nc ) .
measurements 
 of ph were performed on a fisher scientific ap71 ph / mv/c meter 
 with a ph / atc electrode .
determinations of fluoride ion concentration 
 were performed on the same meter with a thermo scientific 9609bn combination 
 fluoride electrode .
large - scale dialyses were performed with thermo 
 scientific slide - a - lyzer dialysis cassettes ( molecular weight cutoff 
 of 10 kda ) unless otherwise specified .
small - scale dialyses were performed 
 with emd chemicals d - tube mini dialyzer ( molecular weight cutoff of 
 1214 kda ) .
radioactivity was determined with a packard tri - carb 
 2100tr liquid scintillation analyzer using perkinelmer ultima gold 
 scintillation fluid .
for centrifugations , a dupont sorvall rc 5b plus 
 centrifuge was used with either an sla-3000 or an sa600 rotor .
enzyme purification 
 was carried out on a ge healthcare life sciences kta fplc 
 system .
the column media used were cm sepharose fast flow for weak 
 cationic exchange mode , deae sepharose fast flow for weak anionic 
 exchange mode , and hydroxylapatite ( ha ) for mixed mode .
a prepacked 
 hiprep sephacryl s-200 high - resolution column was used for size - exclusion 
 mode .
electrophoresis was carried out on a bio - rad mini - protean tetra 
 cell using a vwr accupower model 300 power supply .
lc / ms / ms was performed 
 on thermo fisher q exactive mass spectrometer .
mass spectrometry of 
 intact mass of gaba - at was performed on a velos orbitrap elite ( thermofisher ) . 

crystallographic data were collected on beamline 23 id - b and 23 id - d 
 of gm / ca of the advanced photon source ( aps ) using x - rays of 0.99 
  wavelength and a rayonix ( formerly mar - usa ) 4  4 tiled 
 ccd detector .
all reagents 
 and materials were purchased 
 from sigma - aldrich co. except the following : centrifugal filters ( molecular 
 weight cutoff value of 10 kda and 30 kda ) were purchased form emd 
 millipore ; dowex 50 and sodium dodecyl sulfate were purchased from 
 bio - rad ; [ h]sodium borohydride was purchased from american 
 radiolabeled chemicals , inc . ; pig brains were a generous gift from 
 the park packing co. ( chicago , il ) ; all of the buffers and solvents 
 used for fplc analyses were filtered through ge healthcare 0.45 m 
 nylon membranes .
gabase ( pseudomonas fluorescens ) 
 and succinic semialdehyde were purchased from sigma - aldrich .
gaba - at ( 2.65 mg / ml , specific activity 
 2.1 unit / mg ) was purified from pig brain by the procedure described 
 previously .
succinic semialdehyde dehydrogenase 
 ( ssdh ) was obtained from gabase , a commercially available mixture 
 of ssdh and gaba - at , using a known procedure .
the final assay solution consisted of 11 mm 
 gaba , 1.1 mm nadp , 5.3 mm -kg , 2 mm -mercaptoethanol , 
 and excess ssdh in 50 mm potassium pyrophosphate buffer , ph 8.5 . the 
 change in uv absorbance at a wavelength of 340 nm at 25
c caused 
 by the conversion of nadp to nadph is proportional to 
 the gaba - at activity .
all enzyme assays were recorded with a perkinelmer 
 lambda 10 uv / vis spectrophotometer .
gaba - at ( 2 g ) was incubated with cpp-115 ( 0 , 0.2 , 0.4 , 0.6 , 
 0.8 , 1.0 , 1.2 , 1.4 , 1.6 , 1.8 , and 4 equiv ) at 25 c in 50 mm 
 potassium pyrophosphate buffer solution , containing 2 mm -ketoglutarate 
 and 1 mm -mercaptoethanol .
aliquots ( 2 l ) were withdrawn 
 at timed intervals and were added immediately to the assay solution 
 ( 96 l ) containing excess ssdh ( 2 l ) .
gaba - at ( 60 l ; final concentration 1 m ) 
 was preincubated for 3 h with various amounts of cpp-115 ( 0 , 1 , 1.8 , 
 4 , 40 , and 80 equiv ) in 50 mm pyrophosphate buffer ( ph 8.5 ) containing 
 0.625 mm -ketoglutarate , in a total volume of 160 l 
 at 25 c . in another experiment , it was preincubated for 24 h 
 with various amounts of cpp-115 ( 0 , 1 , 2 , 4 , 40 , 80 , 100 , 1000 , and 
 2000 equiv ) .
after preincubation , the enzyme solutions were transferred 
 to d - tube mini dialyzers and exhaustively dialyzed against buffer 
 ( 50 mm pyrophosphate buffer containing 0.1 mm -ketoglutarate 
 and 0.1 mm plp , ph 8.5 ) at 4 c .
the enzyme activity remaining in each of the 
 solutions was assayed per time interval .
gaba - at ( 150 l ) was incubated in 100 mm 
 potassium pyrophosphate buffer at ph 8.5 , containing 2 mm cpp-115 
 and 2.5 mm -ketoglutarate in a total volume of 510 l . 

the incubation mixture 
 was protected from light and was carried out at room temperature . 

the experiment was repeated with 2.5 mm plp supplemented in the incubation 
 mixture . to construct a standard curve , the relative potential 
 ( mv ) of sodium fluoride solutions in the range of 1 
10 to 5  10 m in total ionic 
 strength adjustment buffer ( tisab ; made from 5.8 g of sodium chloride 
 and 5.7 ml of acetic acid in 100 ml of water , adjusted to ph 5.2 ) 
 was measured .
the measured potential was plotted against the concentration 
 of sodium fluoride . at different incubation time
( 1 , 2 , 5 , 7 , 17 , 
 24 , 27 h ) , each aliquot was removed from the incubation samples and 
 mixed with tisab to measure their relative potentials . using the formula 
 obtained from the standard curve , the mv readings were converted to 
 concentration of fluoride ions in the samples .
the readings from the 
 control sample were subtracted from the inactivated sample , and the 
 concentration was divided by the concentration of gaba - at to obtain 
 the equivalents of fluoride ion released per inactivation event .
the similar experiment 
 was run as above but without -ketoglutarate to test the amount 
 of fluoride released during a one - turnover event .
gaba - at ( 200 l ) 
 was incubated in 100 mm potassium pyrophosphate buffer at ph 8.5 , 
 containing 2 mm cpp-115 in a total volume of 510 l . when there 
 is no -ketoglutarate in the mixture to regenerate plp , the 
 reaction stops at one turnover per active site
thirty drops of 1 m naoh were added to 1.8 
 ml of 0.5 m plp and cooled to 0 c in an ice bath .
the mixture 
 of sodium borohydride ( 5.8 mg , 0.15 mmol ) and [ h]-sodium 
 borohydride ( 25 mci ) in 450 l of 0.1 m naoh was added to the 
 plp solution and stirred for 1 h at 0 c .
after being stirred , 
 120 l of concd hcl was added to the solution slowly ( became 
 ph 4 ) . to the solution at ph 4 ,
20 equiv of ground mno2 was added , and the mixture was stirred at room temperature for a 
 total of 2 h. the mixture was brought to ph 8 with 1 m naoh , and the 
 solution was centrifuged .
the supernatants were collected and were 
 loaded onto a gel filtration column packed with bio - rad ag1-x8 resin 
 ( hydroxide form ) . the mobile phase used was water and 5 m acetic acid 
 ( gradient of 90% water to 0% water ) , flowing at 1.5 ml / min for 300 
 min .
fractions were collected every 10 ml and tested for its uv absorption 
 and radioactivity .
the lyophilized product was redissolved in 100 l of 1 mm plp 
 and loaded onto an hplc with an econosil c18 column ( 10 mm  
 250 mm , 10 m ) .
the mobile phase used was 0.1% aqueous tfa with 
 5% acetonitrile flowing at 0.5 ml / min for 40 min . under these conditions , 

fractions running with the plp peak were collected 
 and counted for radioactivity using liquid scintillation counting . 
 the product was collected and lyophilized .
gaba - at that had been reconstituted with [ h]plp was incubated at room temperature and protected from light 
 in 100 mm potassium phosphate buffer containing cpp-115 ( 2 mm ) , -ketoglutarate 
 ( 3 mm ) , and -mercaptoethanol ( 3 mm ) , in a total volume of 100 
 l at ph 7.4 .
a negative control was run under identical conditions 
 as above , excluding the inactivator .
a positive second control was 
 run with 3 mm gaba in the absence of inactivator and -ketoglutarate . 

the first control should release the cofactor as plp , and the second 
 control should release it as pmp .
after incubation for 18 h , the activity 
 of gaba - at was less than 1% of control , and the solutions were adjusted 
 to ph 11 with 1 m koh and incubated for 1 h. trifluoroacetic acid 
 ( tfa ) was added to quench the base and make the solution 10% v / v tfa . 

the resulting denatured enzyme solution was microcentrifuged for 5 
 min at 10  000 rpm after standing at room temperature for 10 
 min .
a small amount of white solids was seen at the bottom of the 
 tube .
the supernatants were collected individually . to wash the pellets , 
 50 l of 10% tfa
was added to each tube , vortexed , and microcentrifuged 
 for another 5 min .
cofactor analysis was carried out by 
 dissolving the solids obtained from lyophilization with 100 l 
 of a solution containing 2.5 mm plp and 2.5 mm pmp as standards and 
 then injecting the samples into the hplc with an econosil c18 column 
 ( 4.6 mm  150 mm , 10 m ) .
the mobile phase used was 0.1% 
 aqueous tfa flowing at 0.5 ml / min for 25 min .
then the flow rate was 
 increased to 1 ml / min from 25 to 30 min , and then a solvent gradient 
 into 95% acetonitrile was run over the next 30 min . under these conditions , 

fractions were collected every 
 minute , and the radioactivity was measured by liquid scintillation 
 counting .
gaba - at ( 0.3 nmol ) was incubated 
 in 50 mm ammonium bicarbonate buffer ( ph 7.4 ) containing 0.1 mm cpp-115 , 
 1 mm -ketoglutarate , and 0.1 mm plp in a total volume of 70 
 l at room temperature in the dark for 12 h. a control containing 
 everything except cpp-115 was also incubated .
after 12 h , gaba - at 
 in the inactivated sample was less than 1% active vs control .
formic 
 acid ( 1 l ) was added to each reaction mixture , and both were 
 centrifuged in a 0.5 ml 30 kda mwco centrifuge tube ( millipore ) at 
 14000 g for 4 min or until most of the solution had 
 passed through .
an additional 20 l of 50 mm ammonium bicarbonate 
 was added above the filter and centrifuged for 3 min . the flow - through 
 ( 20 ul )
was injected onto a luna c18(2 ) column ( 100 a , 2  150 
 mm , 5 m , phenomenex ) . a 60 min gradient ( agilent 1100 hplc , 
 solvent a = 5% acetonitrile and 0.1% formic acid ; solvent b = 0.1% 
 formic acid in acetonitrile ) was run from 280% b over 40 min . 

the top five most abundant ions in negative ion mode were selected 
 for fragmentation using mass normalized collision energies .
commercially 
 available 3-oxo-1-cyclopentanecarboxylic acid ( sigma ) was injected 
 as a standard for comparison .
three reaction 
 samples were prepared as described previously with only gaba - at , including 
 vigabatrin , or cpp-115 .
after incubation 
 with or without inhibitors , half of each reaction mixture was reduced 
 with 15 mm sodium cyanoborohydride for 1 h. both reduced and nonreduced 
 samples were buffer exchanged into 50 mm ammonium acetate using 30 
 kda mwco filters ( millipore ) .
nano - lc / ms / ms runs were done on a 75 
 m i d  10 cm kinetex c8 ( phenomenex ) column connected 
 to an autosampler ( dionex ultimate 3000 rslcnano system ) and a velos 
 elite orbitrap ( thermofisher ) mass spectrometer .
, gaba - at 
 and cpp-115-inactivated gaba - at were exchanged into a buffer that 
 contained 40 mm sodium acetate ( ph 5.5 ) .
after the initial crystallization 
 screening and optimization , the proteins were crystallized via the 
 hanging drop method .
hanging drops were prepared by mixing 1 l 
 of 12 mg / ml native gaba - at protein solution and 1 l of the 
 reservoir solution , containing 0.1 m ammonium acetate , 0.1 m bis - tris 
 ( ph 5.5 ) , and 17% w / v peg 10  000 .
crystals appeared within 
 24 h at 20 c and grew for 56 days before harvesting . 

crystals with good morphology and large sizes were transferred to 
 cryoprotecting conditions , which contain 20% glycerol in addition 
 to the original composition of the reservoir solution , before being 
 frozen in liquid nitrogen .
the same method of crystallization was 
 applied for cpp-115-inactivated gaba - at ; however , before freezing 
 in liquid nitrogen , the inactivated crystals were transferred to a 
 cryoprotecting solution that contained 20% glycerol and 2 mm cpp-115 , 
 in addition to the compounds of the reservoir solution .
crystallographic 
 data were collected on beamlines 23id - b and 23id - d of gm / ca@aps of 
 the advanced photon source ( aps ) using x - rays of 0.99  wavelength 
 and rayonix ( formerly mar - usa ) 4  4 tiled ccd detector with 
 a 300 mm sensitive area .
data collection and processing statistics 
 are given in table s1 ( supporting information ) .
molecular replacement 
 for the native gaba - at was carried out using the program phaser from ccp4 software 
 suite , using the previously reported coordinates of gaba - at from pig 
 liver ( pdb i d : iohv ) as the starting search model .
the initial rfree and r factor of the correct solution 
 were 0.1904 and 0.1934 , respectively .
manual adjustment and modification of the structure based 
 on electron density maps were performed using the program coot .
the rfree and r factor values of the final model were 0.1513 and 0.1766 , 
 respectively .
the data sets from the inactivated gaba - at crystals 
 were isomorphous to those of native gaba - at .
therefore , rigid body 
 refinement could be used directly , placing the previously refined 
 native gaba - at model into the asymmetric unit of inactivated gaba - at 
 crystals .
model building and refinements of the inactivated structures 
 were carried out following the same protocol as the native structure . 

there were no ligand coordinates included in the refinement until 
 the refinement converged . without ligands built in , the fo 
fc map shows a well - defined 
 electron density supporting the existence of the bound ligand .
the structure of the inactivator was made in the program chemdraw 
 generating a  mol  file as the output .
this output ligand 
 structure was then regularized and its chemical restraints were generated 
 in the program jligand .
using coot , the 
 inactivator was manually fit to the residual electron density in the 
 difference ( fo  fc ) map .
the rfree and r factor for the inactivated structure were 0.1575 and 0.1872 , 
 respectively .
in essence , 
 the ligands ( including the cofactor ) were docked into the active site 
 of the prepared protein using autodock 4.2 , with lys329 being flexible .
the best docked structures were then 
 refined by molecular mechanics , using gromacs 4.5 .
the sequence utilized started with energy minimization , 
 continued with molecular dynamics ( 4 ns ) , and had a final energy minimization 
 step .
 -aminobutyric 
 acid aminotransferase ( gaba - at ) is a pyridoxal 
 5-phosphate ( plp)-dependent enzyme that degrades gaba , the 
 principal inhibitory neurotransmitter in mammalian cells . 
 when the 
 concentration of gaba falls below a threshold level , convulsions can 
 occur . 
 inhibition of gaba - at raises gaba levels in the brain , which 
 can terminate seizures as well as have potential therapeutic applications 
 in treating other neurological disorders , including drug addiction . 
 among the analogues that we previously developed , ( 1s,3s)-3-amino-4-difluoromethylene-1-cyclopentanoic 
 acid ( cpp-115 ) showed 187 times greater potency than that of vigabatrin 
 , 
 a known inactivator of gaba - at and approved drug ( sabril ) for the 
 treatment of infantile spasms and refractory adult epilepsy . 
 recently , 
 cpp-115 was shown to have no adverse effects in a phase i clinical 
 trial . here 
 we report a novel inactivation mechanism for cpp-115 , 
 a mechanism - based inactivator that undergoes gaba - at - catalyzed hydrolysis 
 of the difluoromethylene group to a carboxylic acid with concomitant 
 loss of two fluoride ions and coenzyme conversion to pyridoxamine 
 5-phosphate ( pmp ) . 
 the partition ratio for cpp-115 with gaba - at 
 is about 2000 , releasing cyclopentanone-2,4-dicarboxylate ( 22 ) and two other precursors of this compound ( 20 and 21 ) . 
 time - dependent inactivation occurs by a conformational 
 change induced by the formation of the aldimine of 4-aminocyclopentane-1,3-dicarboxylic 
 acid and pmp ( 20 ) , which disrupts an electrostatic interaction 
 between glu270 and arg445 to form an electrostatic interaction between 
 arg445 and the newly formed carboxylate produced by hydrolysis of 
 the difluoromethylene group in cpp-115 , resulting in a noncovalent , 
 tightly bound complex . 
 this represents a novel mechanism for inactivation 
 of gaba - at and a new approach for the design of mechanism - based inactivators 
 in general . 
Introduction
Results
Discussion
Conclusions
Experimental Procedures