while the number of 
 protein sequences deposited in public databases 
 continues to expand exponentially , determining 
 the function of the encoded proteins remains slow . except where sequence 
 identity to a protein of known function is high , the activity of a 
 newly sequenced protein must be interrogated with candidate ligands 
 or substrates . this can be done empirically , by screening for binding 
 or substrate turnover or by a mixture of computational prediction , for 
 instance by docking molecular libraries and subsequent experimental testing . both 
 approaches rely on screening libraries of small molecules , such as 
 metabolites . if the right metabolite , 
 or a close analogue , is present in the library , it may be detected 
 as substrate , whereas if it is not , either no activity will be assigned 
 or it may be mis - assigned . in the latter case , however , the multiple chemotypes 
 present in biological small molecules , and their exponential scaling 
 when combined into more complex biological compounds , make full coverage 
 of biorelevant chemical space difficult to ensure . in drug discovery , the combinatorial explosion of chemotypes with 
 molecular size has been addressed by screening libraries of fragment 
 molecules . because fragments are smaller 
 than druglike molecules ( typically less than 17 non - hydrogen atoms ) , 
 fragment chemical space is about 50 orders - of - magnitude smaller than 
 druglike chemical space , enabling fragment 
 libraries to cover chemical space better than libraries of more complex 
 molecules . individual fragment inhibitors 
 usually present simple chemotypes that are only expanded out to fully 
 elaborated molecules after initial hits are discovered ; this has been 
 a remarkably successful approach . a fragment - based strategy could be an attractive alternative 
 to 
 the full enumeration of metabolite space for substrate discovery . 
 not only would it cover potential substrate space far more efficiently , 
 but it would also increase the number of representative molecules 
 that can be physically sourced ; currently , many known metabolites 
 and biogenic molecules are simply unavailable for testing this is 
 far less of a problem for fragments , where molecules containing core 
 reactant groups are readily available ; for instance , over 700,000 
 accessible fragments are cataloged in the zinc database . a key question is whether a substrate , stripped 
 to the core reactive group on which the catalytic machinery of an 
 enzyme acts , retains enough recognition elements to be an effective , 
 or at least a detectable , enzyme substrate . it could be that enzyme 
 catalysis is so demanding that most of the atoms of the substrate 
 must be engaged with the enzyme before catalysis will occur . several 
 lines of evidence support this view , including studies showing that 
 fragmentation of cytidine into component fragments lowered the activity 
 for cytidine deaminase by 49 orders - of - magnitude and that fragmentation of a transition - state 
 analogue of calf adenosine deaminase led to losses of up to 6 orders - of - magnitude 
 in affinity . also , as shown by jencks , there is no reason why the binding energies of component fragments 
 should sum up to the binding or catalytic activity of a full substrate , 
 owing to the nonadditive , nonequilibrium effects of chemical connectivity . conversely , other studies suggest that fragments can be built up additively 
 for affinity and catalytic recognition . for instance , the well - studied 
 enzyme chymotrypsin hydrolyzes a variety of substrates , including p - nitrophenyl acetate and p - nitroanilides . these compounds only share a very reduced fragment at the reaction 
 center ( two common heavy atoms : c(=o ) ) , which would argue in 
 favor of a fragment - based approach . it has also been possible to deconstruct 
 at least some substrates and transition - state analogues into component 
 fragments . for example , the transition - state analogue immucillin - h , 
 which inhibits purine nucleoside phosphorylase with a ki of 28 pm , was deconstructed into purine and iminoribitol 
 with a loss of only 30-fold in the multiplied affinities of the component 
 fragments . crystallographic studies confirmed 
 that the geometry of these fragments , when bound to the enzyme , recapitulates 
 that of the entire immucillin - h . fragments 
 of the thymidylate synthase substrate deoxyuridine monophosphate , 
 2-deoxyuridine , and 2,5-dideoxyuridine , also 
 conserve their binding mode when crystallized in the presence of phosphate , 
 although smaller fragments are either poorly ordered in the active 
 site or bind nonspecifically . more recently , 
 the substrate specificity of the mis - annotated enzyme atu3266 from agrobacterium tumefaciens c58 was determined , starting 
 from a very weak fragment hit ( kcat / km = 4 m s ) and resulting in a potent substrate ( kcat / km = 2.8  10 m s ) . similarly , for triosephosphate isomerase , the difference in activation 
 barrier for the isomerization of whole substrate and substrate in 
 pieces is large ( 6.6 kcal / mol ) but product formation is still detectable . large increases in proteolytic activity also 
 have been observed when long - chain substrates are hydrolyzed by pepsin 
 and elastase . lastly , in addition to the successes 
 in stepwise optimization of fragment inhibitors for drug discovery , a fragment - based approach has been used to develop enzyme inhibitors , 
 based on initial substrate turnover . in short , 
 there is evidence to both support and undermine the use of fragments 
 for substrate discovery . the potential benefit of fragment screens 
 against genomic targets , which may dramatically expand our ability 
 to probe chemotype space , spurred us to explore this question further . if it is true that fragments can be used as probe substrates , fragments 
 of extant substrates should retain substantial turnover , assuming 
 that they keep the reactive center intact . here we explore this idea 
 systematically on the substrates of six enzymes from three different 
 enzyme superfamilies : the amidohydrolase superfamily members adenosine 
 deaminase ( ada ) , 5-methylthioadenosine / s - adenosyl - homocysteine 
 deaminase ( sahd ) , and isoaspartyl dipeptidase ( iad ) ; the haloacid 
 dehalogenase superfamily members phosphoserine phosphatase ( psp ) and 
 flavin mononucleotide phosphatase ( fmp ) ; and ampc -lactamase 
 ( ampc ) . substrates for each enzyme were deconstructed into fragments 
 ( figure 1 ) , which contained either the catalytic 
 core of the full - length substrate , or  side - chains  
 of the substrate not directly involved in catalysis . the fragments 
 were tested for enzyme turnover or enzyme inhibition , typically one 
 at a time . in almost every case , the resulting fragments several 
 variants of which were tried for each enzyme  showed a drop 
 of 10 or more in kcat / km relative to the canonical substrate , and for 
 most compounds binding could not be detected . these results have implications 
 for our ability to extend the fragment approach , so successful for 
 inhibitor discovery , to substrate discovery , and may illuminate the 
 nature of the small - molecule environment against which enzymes have 
 evolved . multiple ways of fragmenting the substrates of adenosine deaminase 
 ( ada ) ; mta / sah deaminase ( sahd ) ; phosphoserine phosphatase ( psp ) ; 
 flavin mononucleotide phosphatase ( fmp ) ; and ampc -lactamase 
 ( ampc ) . only one fragment was typically tested at a time . for isoaspartyl 
 dipeptidase ( iad ) , recognition of the component amino - acid monomers 
 was investigated . the canonical substrate of ada , adenosine 
 ( 1 ) , was fragmented in stages , beginning with the removal 
 of the hydroxyl groups at c2 , c3 , and c5 of 
 the molecule ( 2 , 3 , 4 ) , and 
 with the dideoxy derivative , 2,5-dideoxy adenosine 
 ( 5 ) . subsequently , the entire ribose was removed to give 
 9-hydroxymethyladenine ( 6 ) , 9-methyladenine ( 7 ) , and adenine ( 8) ; all retained the core reactive purine 
 amine on which the deamination reaction occurs . value for 
 adenosine is 7.6  10 m s. while the 2-deoxy derivative 2 retained most of that activity ( kcat / km 4.1  10 m s ) , the 3-deoxy 3 lost 
 over 1 order of magnitude of activity ( kcat /km 2  10 m s ) , and in the 5-deoxy and dideoxy analogues 4 and 5 no activity could be detected . for the 
 adenine derivatives 6 , 7 , and 8 , kcat / km values were reduced by 5 orders of magnitude ( table 1 ) . it may be that , while purines themselves 
 are poor substrates , adding a purine and a ribose fragment simultaneously 
 to the enzyme would enhance activity . to investigate this , adenine 
 ( 8) was added to the enzyme which had been preincubated 
 with 1-deoxy - d - ribose ( 9 ) ( 5 mm ) , but correspondingly , deamination 
 of adenosine ( 1 ) was not inhibited by addition of either 9 or ribosylamine ( 10 ) at concentrations up to 
 6.5 mm , indicating that these two fragments do not bind detectably 
 to the active site of the enzyme . to explore the specificity of ada , 
 cytosine ( 11 ) and guanine ( 12 ) were tested 
 as substrates ; neither showed detectable activity . sahd deaminates 5-methyl - thioadenosine ( 13 ) , s - adenosyl - homocysteine ( 14 ) and adenosine 
 ( 1 ) with kcat /km values of 5  10 m s , 5.8  10 m s , and 9.2  10 m s , respectively ( table 2 ) . this enzyme is mechanistically related 
 to ada , but their sequence identity is low ( 22% ) ; these two enzymes 
 fall into two different clades of the amidohydrolase superfamily and 
 are from different clusters of orthologous groups . purine fragments that retain the reactive core of the larger 
 substrates were tested for activity . no deamination could be detected 
 for 9-hydroxymethyladenine ( 6 ) , 9-methyladenine ( 7 ) , and adenine ( 8) after incubating these compounds 
 ( 0.1 mm ) for 12 h with 0.001 mm enzyme ( kcat / km < 2.3 m s ) ( table 2 ) . to further probe 
 binding , the ribose fragments 1-deoxy - d - ribose ( 9 ) and ribosyl amine ( 10 ) , and methionine ( 15 ) were tested as inhibitors of sahd using 13 as a substrate . 
 at concentrations of 5.0 mm , 9 , 10 , and 15 did not inhibit the enzyme detectably ( ki > 5 mm ) . we note that the crystal structure of sahd , 
 determined in the presence of a high concentration of methionine ( 15 ) as an antioxidant , demonstrates that this fragment can 
 bind to the protein in the absence of other substrates . to investigate a possible synergistic role of 15 , the enzyme was preincubated with this fragment ( up to 
 10 mm ) before addition of 8 , but still no deamination 
 was observed . thus , for mta - sah deaminase , purine fragments of the 
 canonical substrate , even though they retain the core adenine reactive 
 center , lose over 5 orders of magnitude of activity relative to the 
 natural substrate of the enzyme . iad hydrolyzes several -aspartyl 
 peptides , of which -aspartyl - leucine 
 ( 16 ) is the best recognized , with a kcat / km of 10 m s. we were interested 
 in determining if the enzyme would recognize any of the component 
 amino acid monomers , even as inhibitors . accordingly , iad was assayed 
 with 0.5 mm of the substrate 16 in the presence and absence 
 of 2 mm concentrations of the following l - amino acids : leucine 
 ( 17 ) , alanine ( 18 ) , glutamate ( 19 ) , phenylalanine ( 20 ) , glycine ( 21 ) , histidine 
 ( 22 ) , isoleucine ( 23 ) , lysine ( 24 ) , methionine ( 15 ) , asparagine ( 25 ) , proline 
 ( 26 ) , glutamine ( 27 ) , arginine ( 28 ) , serine ( 29 ) , threonine ( 30 ) , valine 
 ( 31 ) , tryptophan ( 32 ) , and tyrosine ( 33 ) ( table s1 supporting information [ si ] ) . no measurable inhibition was observed for any of these amino 
 acids ( ki < 4 mm ) . psp dephosphorylates phosphoserine ( 34 ) with a kcat / km of 3.6  10 m s and phosphothreonine ( 35 ) with a kcat /km of 3  
 10 m s ( table 3 ) . here too , the substrates are small to begin with , 
 but a reasonable fragmentation was tested using the decarboxy analogue 
 of phosphoserine , ethanolamine phosphate ( 36 ) . this perturbation 
 was sufficient to reduce activity on this molecule , relative to phosphoserine , 
 by 5 orders of magnitude , to a kcat / km of 4.2 m s. meanwhile , serine ( 29 ) itself is a weak competitive 
 inhibitor with a ki of 0.5 mm . fmp catalyzes 
 the dephosphorylation of flavin mononucleotide ( 37 ) with 
 a kcat / km of 
 2.4  10 m s ( table 4 ) . fragments where the flavin ring 
 was removed , leaving sugar phosphate analogues , were tested as substrates . mannose-1-phosphate ( 38 ) , glucose-1-phosphate ( 39 ) and ribose-5-phosphate ( 40 ) , though far less 
 active as substrates than flavin mononucleotide , do retain substantial 
 activity , with kcat / km of 2  10 m s , 2.6  10 m s , and 2.1  10 m s , respectively . the fragments lumichrome 
 ( 41 ) , mannose ( 42 ) , and glucose ( 43 ) showed no measurable inhibition of fmp - catalyzed dephosphorylation 
 of 38 ( ki values > 0.5 , 
 20 , 
 and 20 mm , respectively ) . unfortunately , investigating a possible 
 synergistic effect of 41 on the activity of 38 , 39 , and 40 was not possible due to technical 
 issues ( fluorescence and background phosphate interference , see methods 
 in si ) . ampc catalyzes the hydrolysis of cephalothin 
 ( 44 ) with a kcat / km of 7  10 m s ( table 5 ) . we began 
 with fragments that retained the -lactam ring , on which hydrolysis 
 takes place , but lacked most of the side chains characteristic of 
 -lactam antibiotics . starting with the minimal bicyclic cephalosporin 
 ring system ( 45 ) and building up through 46 to cephalosporanic acid ( 47 ) itself , no measurable 
 activity was observed . only for 7-n - formyl - cephalosporanic 
 acid ( 48 ) , which almost entirely recapitulates cephalothin , 
 is measurable activity achieved , and then it is almost entirely restored 
 with a kcat /km of 2.9  10 m s ( figure s1 in the si ) . thus , the addition 
 of a single formyl moiety , distal to the catalytic center , restores 
 5 log - orders of activity to the cephalosporin family . correspondingly , 
 even binding for the smaller fragments 46 and 47 was difficult to detect , with ki values 
 of about 2 and 5 mm , respectively . though the amidation of the n7 
 was sufficient to confer activity , the full thiophene acetamide fragment 49 did not detectably inhibit the enzyme ( ki > 20 mm ) , nor did the methyl acetate fragment 50 , representing the right - hand side of cephalothin ( ki > 20 mm ) . the structures 
 of three cephalothin 
 fragments , 45 , 46 , and 48 in 
 complex with ampc were determined by x - ray crystallography , with resolutions 
 ranging from 1.37 to 1.71  . fc difference electron density maps generated early in 
 refinement ( figure s2 and table s2 in the si ) , allowing reliable model building of the enzyme electron density for 48 in the ampc active site is unambiguous in both monomers , 
 with a clear covalent bond between ser64 and the fragment substrate , 
 as expected ( figure 2a and figure s2 in the si ) . the hydrolysis of the beta - lactam ring results 
 in a carbonyl oxygen that interacts with s64 and a318 backbone nitrogens 
 in the oxyanion hole of the enzyme . this 
 structure thus captures the stable acyl - enzyme intermediate step between 
 the transition - state acylation and deacylation complexes . ( a ) the structure of ampc in complex with 48 shows a covalent bond between ser64 and the fragment substrate , 
 ( see also figure s2 in the si ) and captures 
 the stable acyl - enzyme intermediate step between the transition state 
 acylation and deacylation complexes . ( b ) the structure of ampc in 
 complex with 45 shows the fragment in its product form , 
 bound in two orientations to a distal subsite of the large overall 
 binding site , interacting with ser212 , tyr221 , and gly320 . although we could 
 detect neither turnover of 45 nor binding to the enzyme , 
 we were able to observe this compound in the hydrolyzed form in both 
 monomers ( figure 2b and figure s2 in the si ) , via x - ray crystallography after a 10 min 
 soak at 100 mm . the hydrolyzed form of 45 does not bind 
 to the catalytic site , but rather to a distal subsite of the large 
 binding site , interacting with ser212 , tyr221 , and gly320 . two orientations 
 of the ligand are observed , but in both the carboxylates hydrogen 
 bond with the backbone nitrogens of ser212 and gly320 , as previously 
 observed for anionic ligands targeting this site . it may be that this orientation represents an intermediate step 
 in the exit of the product in ampc  the contacting residues 
 are well - conserved , even though ser212 and gly320 rarely interact 
 with the better cephalosporin substrates of the enzyme . while 46 was also inert to 
 catalysis by ampc , this compound did weakly inhibit the enzyme . in 
 the crystal structure of the ampc/46 complex , 46 is bound at the surface of the protein , away from the active site , 
 in its hydrolyzed form ( figure s2 in the si ) . intriguingly , the substrate cephalotin was previously observed 
 to bind to the same site , also in the hydrolyzed form . neither the complex with 46 nor 45 supports a catalytically competent recognition of fragment 
 substrates by -lactamase , though both contain the core cephalosporin 
 ring . the appearance of this hydrolysis product in the structure may 
 reflect slow turnover by the enzyme , over the course of the crystallization 
 experiment , but we can not exclude the possibility that the hydrolysis 
 product reflects a background reaction in solvent , to which -lactams 
 are prone . a key observation from 
 this study is that substrate fragments for 
 five of the six enzymes lost essentially all measurable activity or 
 binding , with a reduction of at least 56 orders of magnitude 
 relative to that of the canonical substrate . detectable activity was 
 not regained until most of the substrate had been recapitulated , typically 
 by adding back recognition elements far from the reactive center ( figure 3 ) . clipping only two or three heavy atoms , well 
 removed from the reactive site , was sufficient to almost completely 
 oblate enzymatic activity . for instance , removing a single hydroxyl 
 from adenosine ( 1 ) to form 2- or 3-deoxyadenosine 
 ( 2 and 3 ) reduces the activity of adenosine 
 deaminase by 10-fold ( table 1 ) , as does eliminating 
 the thiophene ring from cephalothin for -lactamase ( table 5 ) . removing one additional hydroxyl moiety from 2 to form 2,5-dideoxy adenosine ( 5 ) completely abolishes the adenosine deaminase activity , while removing 
 the formyl group from 48 eliminates detectable ampc activity . 
 molecules representing the core of the substrate , containing the key 
 reactive group , and representing over 70% of its atoms and functionality , 
 typically had little or no measurable activity against their respective 
 enzymes ( figure 3 ) . moreover , most showed no 
 detectable enzyme inhibition . whereas fragment - based approaches are 
 sometimes suitable for substrate discovery , as observed earlier for 
 purine nucleoside phosphorylase and atu3266 , they seem unreliable as a general strategy . plot of 
 % compound activity ( as compared to the entire substrate ) 
 as a function of size . the high sensitivity of substrate 
 reactivity to small chemical 
 insults seems to contrast with successful efforts to use fragment 
 substrates as inhibitor leads and with efforts to 
 additively improve the affinity of inhibitory fragments . peptide- 
 and phosphate - bearing fragments have been used as substrate leads 
 for proteases and phosphatases with great success . even in that work , however , substrate recognition was often decreased 
 by 5 orders of magnitude compared to that of canonical substrates . this activity loss can be accepted when one knows the fundamental 
 activity for which one is probing ; it is harder to tolerate when seeking 
 to identify the core enzyme activity from a screen of a diverse library 
 of potential substrates . it is challenging to reconcile the 
 large effects on substrate recognition 
 with those of inhibitors , where it is uncommon to find that removal 
 of a single atom or small moiety reduces affinity by 79 kcal / mol , 
 as was observed here . not only did small changes in the substrate 
 have pronounced effects , the residual pieces often did not measurably 
 bind to the enzymes . at least partly , this may reflect the limits 
 of detection over background , and more importantly the compounding 
 effects of reduced substrate recognition on catalytic competence . 
 because substrate binding is usually weak , a loss of 3 kcal / mol in 
 substrate affinity will not only reduce turnover by 100-fold ( in the kcat / km regime ) but 
 may also ramify through poor substrate alignment to a lower kcat . however , it also seems that , for many enzymes , 
 there is a substantial 
 nonadditivity to substrate recognition in catalysis . thus , functional 
 groups that contribute substantially to recognition when presented 
 as part of the full substrate , are not recognized in substrate fragments . 
 for inhibitors , conversely , recognition is often more additive . the 
 idea of integrated , nonadditive recognition of substrates has precedence 
 in experimental and theoretical enzymology  returning 
 to jencks idea that groups distal to the reactive center may 
 provide crucial binding energy for catalytic recognition  and these results 
 may have implications for promiscuity on the substrate 
 side of enzyme  substrate interactions , providing a counterpoint 
 to the promiscuity of enzyme recognition and evolution . enzyme promiscuity and moonlighting 
 activities seem central to the evolution 
 of new activities , and noncanonical substrates and ligands may be 
 specifically recognized . what this study 
 suggests is that enzyme recognition of optimal substrates for any 
 given reaction is mostly punctate , tolerating little diminution in 
 substrate functional groups . physically , this may reflect the integrated 
 chemistry of small molecules , where each atom contributes to the electronic 
 properties of the entire substrate . the sharp dependence on substrate 
 structure may also reflect specificity constraints faced by enzymes 
 confronted with multiple related metabolites , upon whose product multiple 
 downstream enzymes depend . in such a system , it is possible to duplicate 
 and subsequently mutate an enzyme without detrimental effect on other 
 dependencies , exploiting intrinsic enzyme promiscuity . conversely , 
 changes in the substrate or the product could have cascading effects 
 on the larger cellular system . thus , while enzymes remain plastic 
 to evolution overall , and may be promiscuous with unrelated substrates , they may be selected for high fidelity around the structure of their 
 cognate substrate , in which little change is tolerated ; this idea 
 has also been suggested for signaling molecules and their receptors . that was observed here for flavin 
 nucleotide phosphatase , and has been observed previously for purine 
 nucleoside phosphorylase . also , one can always 
 imagine larger substrates than the core molecules we have investigated . for instance , there are -lactamase substrates that are substantially 
 larger than cephalothin , and for these the removal of functional groups 
 to yield a substrate just below the size of cephalothin only marginally 
 affects their reactivity to -lactamase . however , the larger side chains of these molecules do not 
 make strong interactions with -lactamase , and these may be seen as extraneous groups that are not 
 germane to substrate recognition by the enzyme . the tolerance to diversity 
 of substrate sizes may be more common for a detoxification enzyme 
 like -lactamase , which operates by itself and not in a pathway . 
 whereas we suspect that in most cases substrates do not tolerate fragmenting , 
 and fragments will not be a pragmatic general strategy for function 
 discovery , there will be cases where such a strategy may well be fruitful . for instance , phosphoserine phosphatase only 
 recognizes phosphoserine and phosphothreonine , whereas other phosphatases 
 such as alkaline phosphatase are able to hydrolyze a large range of 
 monophosphate substrates . such broad - specificity 
 enzymes are expected to be less sensitive to small chemical changes 
 in their substrates and to lend themselves more easily to substrate 
 fragmentation . finally , using substrate fragments as probes for new 
 inhibitors and fragments as leads for inhibitor 
 discovery is unaffected by this study ; these approaches 
 to inhibitor discovery continue to hold great promise . most substrate functional groups may be essential for recognition 
 and turnover by a cognate enzyme , and removal of even a small portion 
 of the substrate , distal to the reactive center , can dramatically 
 reduce the activity . once deconstructed into smaller parts , the resulting 
 fragments show little to no activity , and often do not measurably 
 bind to the enzyme active site . enzymes typically did not evolve for 
 inhibitor binding as they did for substrates , and fidelity requirements 
 appear much higher for the latter . this may reflect a need to engage 
 a full substrate to drive reactivity , as distal functional groups 
 can contribute critically to stabilization of a transition state . the idea that the stringent specificity constraints 
 on substrates , as opposed to inhibitors , reflects downstream affects 
 of products on the cell is more speculative . even here , one can imagine 
 testable implications , as designed enzymes , not evolved against pathway 
 constraints , may exhibit broader promiscuity against related substrates 
 and fragments than do natural enzymes . pragmatically , this study supports 
 programs to synthesize and collect a relatively full repertoire of 
 the core metabolites recognized by biology with which to interrogate 
 protein function . this space , though far larger than one would prefer , 
 is not , in the end , unbounded . isoaspartyl dipeptidase 
 ( iad ) and ampc -lactamase ( ampc ) were expressed and purified 
 as previously described . for adenosine deaminase 
 ( ada ) , mta - sah deaminase ( sahd ) , phosphoserine phosphatase ( psp ) and 
 flavin mononucleotide phosphatase ( fmp ) expression and purification , 
 see methods in the si . the hydrolysis rates and inhibition constants 
 for adenosine deaminase and mta - sah deaminase activities were monitored 
 spectrophotometrically in a direct assay . inhibition of isoaspartyl 
 dipeptidase was measured in a spectrophotometric assay coupling the 
 formation of aspartate to the oxidation of nadh . the substrate hydrolysis 
 rates and inhibition constants for phosphoserine phosphatase were 
 measured spectrophotometrically using the enzchek kit ( invitrogen ) . the hydrolysis rates and inhibition constants for flavin mononucleotide 
 phosphatase were monitored using either the enzchek kit or biomol 
 green ( enzo life sciences ) . the hydrolysis of ampc -lactamase 
 substrates and enzyme inhibition were monitored spectrophotometrically 
 ( methods in the si ) . ampc -lactamase 
 structures in complex with 45 , 46 , and 48 were obtained by soaking the crystals in the respective 
 ligand solution . structures were determined between 1.37 and 1.71 
  resolution , and the phases for all structures were determined 
 by difference fourier methods ( methods in the si ) .
predicting substrates for enzymes 
 of unknown function is a major 
 postgenomic challenge . 
 substrate discovery , like inhibitor discovery , 
 is constrained by our ability to explore chemotypes ; it would be expanded 
 by orders of magnitude if reactive sites could be probed with fragments 
 rather than fully elaborated substrates , as is done for inhibitor 
 discovery . to explore the feasibility of this approach 
 , substrates 
 of six enzymes from three different superfamilies were deconstructed 
 into 41 overlapping fragments that were tested for activity or binding . 
 surprisingly , even those fragments containing the key reactive group 
 had little activity , and most fragments did not bind measurably , until 
 they captured most of the substrate features . removing a single atom 
 from a recognized substrate could often reduce catalytic recognition 
 by 6 log - orders . to explore recognition at atomic resolution , 
 the 
 structures of three fragment complexes of the -lactamase substrate 
 cephalothin were determined by x - ray crystallography . 
 substrate discovery 
 may be difficult to reduce to the fragment level , with implications 
 for function discovery and for the tolerance of enzymes to metabolite 
 promiscuity . 
 pragmatically , this study supports the development of 
 libraries of fully elaborated metabolites as probes for enzyme function , 
 which currently do not exist .