Tyr115, gln165 and trp209 contribute to the 1, 2-epoxy-3-(p-nitrophenoxy)propane-conjugating activity of glutathione S-transferase cGSTM1-1

We investigated the epoxidase activity of a class mu glutathione S-transferase (cGSTM1-1), using 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) as substrate. Trp209 on the C-terminal tail, Arg107 on the alpha4 helix, Asp161 and Gln165 on the alpha6 helix of cGSTM1-1 were selected for mutagenesis and kinetic studies. A hydrophobic side-chain at residue 209 is needed for the epoxidase activity of cGSTM1-1. Replacing Trp209 with histidine, isoleucine or proline resulted in a fivefold to 28-fold decrease in the k(cat)(app) of the enzyme, while a modest 25 % decrease in the k(cat)(app) was observed for the W209F mutant. The rGSTM1-1 enzyme has serine at the correponding position. The k(cat)(app) of the S209W mutant is 2. 5-fold higher than that of the wild-type rGSTM1-1. A charged residue is needed at position 107 of cGSTM1-1. The K(m)(app)(GSH) of the R107L mutant is 38-fold lower than that of the wild-type enzyme. On the contrary, the R107E mutant has a K(m)(app)(GSH) and a k(cat)(app) that are 11-fold and 35 % lower than those of the wild-type cGSTM1-1. The substitutions of Gln165 with Glu or Leu have minimal effect on the affinity of the mutants towards GSH or EPNP. However, a discernible reduction in k(cat)(app) was observed. Asp161 is involved in maintaining the structural integrity of the enzyme. The K(m)(app)(GSH) of the D161L mutant is 616-fold higher than that of the wild-type enzyme. In the hydrogen/deuterium exchange experiments, this mutant has the highest level of deuteration among all the proteins tested.We also elucidated the structure of cGSTM1-1 co-crystallized with the glutathionyl-conjugated 1, 2-epoxy-3-(p-nitrophenoxy)propane (EPNP) at 2.8 A resolution. The product found in the active site was 1-hydroxy-2-(S-glutathionyl)-3-(p-nitrophenoxy)propane, instead of the conventional 2-hydroxy isomer. The EPNP moiety orients towards Arg107 and Gln165 in dimer AB, and protrudes into a hydrophobic region formed by the loop connecting beta1 and alpha1 and part of the C-terminal tail in dimer CD. The phenoxyl ring forms strong ring stacking with the Trp209 side-chain in dimer CD. We hypothesize that these two conformations represent the EPNP moiety close to the initial and final stages of the reaction mechanism, respectively.     

Tyrosine residues serve as proton donor in the catalytic mechanism of epoxide hydrolase from Agrobacterium radiobacter

Epoxide hydrolase from Agrobacterium radiobacter catalyzes the hydrolysis of epoxides to their diols via an alkyl-enzyme intermediate. The recently solved X-ray structure of the enzyme shows that two tyrosine residues (Tyr152 and Tyr215) are positioned close to the nucleophile Asp107 in such a way that they can serve as proton donor in the alkylation reaction step. The role of these tyrosines, which are conserved in other epoxide hydrolases, was studied by site-directed mutagenesis. Mutation of Tyr215 to Phe and Ala and mutation of Tyr152 to Phe resulted in mutant enzymes of which the k(cat) values were only 2-4-fold lower than for wild-type enzyme, whereas the K(m) values for the (R)-enantiomers of styrene oxide and p-nitrostyrene oxide were 3 orders of magnitude higher than the K(m) values of wild-type enzyme, showing that the alkylation half-reaction is severely affected by the mutations. Pre-steady-state analysis of the conversion of (R)-styrene oxide by the Y215F and Y215A mutants showed that the 1000-fold elevated K(m) values were mainly caused by a 15-40-fold increase in K(S) and a 20-fold reduction in the rate of alkylation. The rates of hydrolysis of the alkyl-enzyme intermediates were not significantly affected by the mutations. The double mutant Y152F+Y215F showed only a low residual activity for (R)-styrene oxide, with a k(cat)/K(m) value that was 6 orders of magnitude lower than with wild-type enzyme and 3 orders of magnitude lower than with the single tyrosine mutants. This indicates that the effects of the mutations were cumulative. The side chain of Gln134 is positioned in the active site of the X-ray structure of epoxide hydrolase. Mutation of Gln134 to Ala resulted in an active enzyme with slightly altered steady-state kinetic parameters compared to wild-type enzyme, indicating that Gln134 is not essential for catalysis and that the side chain of Gln134 mimics bound substrate. Based upon this observation, the inhibitory potential of various unsubstituted amides was tested, resulting in the identification of phenylacetamide as a competitive inhibitor with an inhibition constant of 30 microM.     

Contribution of Gln9 and Phe80 to substrate binding in ribonuclease MC1 from bitter gourd seeds.

Ribonuclease MC1 (RNase MC1) isolated from bitter gourd (Momordica charantia) seeds specifically cleaves phosphodiester bonds on the 5'-side of uridine. The crystal structures of RNase MC1 in complex with 2'-UMP or 3'-UMP reveal that Gln9, Asn71, Leu73, and Phe80 are involved in uridine binding by hydrogen bonding and hydrophobic interactions [Suzuki et al. (2000) Biochem. Biophys. Res. Commun. 275, 572-576]. To evaluate the contribution of Gln9 and Phe80 to uridine binding, Gln9 was replaced with Ala, Phe, Glu, or His, and Phe80 with Ala by site-directed mutagenesis. The kinetic properties of the resulting mutant enzymes were characterized using cytidylyl-3',5'-uridine (CpU) as a substrate. The mutant Q9A exhibited a 3.7-fold increased K(m) and 27.6-fold decreased k(cat), while three other mutations, Q9F, Q9E, and Q9H, predominantly affected the k(cat) value. Replacing Phe80 with Ala drastically reduced the catalytic efficiency (k(cat)/K(m)) with a minimum K(m) value equal to 8 mM. It was further found that the hydrolytic activities of the mutants toward cytidine-2',3'-cyclic monophosphate (cCMP) were reduced. These results demonstrate that Gln9 and Phe80 play essential roles not only in uridine binding but also in hydrolytic activity. Moreover, we produced double Ala substituted mutants at Gln9, Asn71, Leu73, and Phe80, and compared their kinetic properties with those of the corresponding single mutants. The results suggest that these four residues may contribute to uridine binding in a mutually independent manner.     

Evaluation of peptide-aldehyde inhibitors using R188I mutant of SARS 3CL protease as a proteolysis-resistant mutant

The 3C-like (3CL) protease of the severe acute respiratory syndrome (SARS) coronavirus is a key enzyme for the virus maturation. We found for the first time that the mature SARS 3CL protease is subject to degradation at 188Arg/189Gln. Replacing Arg with Ile at position 188 rendered the protease resistant to proteolysis. The R188I mutant digested a conserved undecapeptide substrate with a K(m) of 33.8 microM and k(cat) of 4753 s(-1). Compared with the value reported for the mature protease containing a C-terminal His-tag, the relative activity of the mutant was nearly 10(6). Novel peptide-aldehyde derivatives containing a side-chain-protected C-terminal Gln efficiently inhibited the catalytic activity of the R188I mutant. The results indicated for the first time that the tetrapeptide sequence is enough for inhibitory activities of peptide-aldehyde derivatives.     

Site-directed mutagenesis of active site residues of phosphite dehydrogenase

Phosphite dehydrogenase (PTDH) catalyzes the unusual oxidation of phosphite to phosphate with the concomitant reduction of NAD(+) to NADH. PTDH shares significant amino acid sequence similarity with D-hydroxy acid dehydrogenases (DHs), including strongly conserved catalytic residues His292, Glu266, and Arg237. Site-directed mutagenesis studies corroborate the essential role of His292 as all mutants of this residue were completely inactive. Histidine-selective inactivation studies with diethyl pyrocarbonate provide further evidence regarding the importance of His292. This residue is most likely the active site base that deprotonates the water nucleophile. Kinetic analysis of mutants in which Arg237 was changed to Leu, Lys, His, and Gln revealed that Arg237 is involved in substrate binding. These results agree with the typical role of this residue in D-hydroxy acid DHs. However, Glu266 does not play the typical role of increasing the pK(a) of His292 to enhance substrate binding and catalysis as the Glu266Gln mutant displayed an increased k(cat) and unchanged pH-rate profile compared to those of wild-type PTDH. The role of Glu266 is likely the positioning of His292 and Arg237 with which it forms hydrogen bonds in a homology model. Homology modeling suggests that Lys76 may also be involved in substrate binding, and this postulate is supported by mutagenesis studies. All mutants of Lys76 display reduced activity with large effects on the K(m) for phosphite, and Lys76Cys could be chemically rescued by alkylation with 2-bromoethylamine. Whereas a positively charged residue is absolutely essential for activity at the position of Arg237, Lys76 mutants that lacked a positively charged side chain still had activity, indicating that it is less important for binding and catalysis. These results highlight the versatility of nature's catalytic scaffolds, as a common framework with modest changes allows PTDH to catalyze its unusual nucleophilic displacement reaction and d-hydroxy acid DHs to oxidize alcohols to ketones.     

Site-directed mutageneses of rat liver cytochrome P-450d: catalytic activities toward benzphetamine and 7-ethoxycoumarin

Catalytic activities toward benzphetamine and 7-ethoxycoumarin of 11 distal mutants, 9 proximal mutants, and 3 aromatic mutants of rat liver cytochrome P-450d were studied. A distal mutant Thr319Ala was not catalytically active toward benzphetamine, while this mutant retained activity toward 7-ethoxycoumarin. Distal mutants Gly316Glu, Thr319Ala, and Thr322Ala displayed higher activities (kcat/Km) toward 7-ethoxycoumarin that were 2.4-4.7-fold higher than that of the wild-type enzyme. Although kcat/Km values of four multiple distal mutants toward benzphetamine were less than half that of the wild type, activities of these mutants toward 7-ethoxycoumarin were almost the same as or higher than the wild-type activity toward this substrate. The distal double mutant Glu318Asp, Phe325Tyr showed 6-fold higher activity than the wild-type P-450d toward 7-ethoxycoumarin. Activities of the proximal mutants Lys453Glu and Arg455Gly toward both substrates were much lower (less than one-seventh) than the corresponding wild-type activities. Catalytic activities of three aromatic mutants, Phe425Leu, Pro427Leu, and Phe430Leu, toward benzphetamine were less than 7% of that of the wild type, while the activities of these aromatic mutants toward 7-ethoxycoumarin were more than 2.5 times higher than the wild-type activity toward this substrate. From these findings, in conjunction with a molecular model for P-450d, we suggest that (1) the relative importance to catalysis of various distal helix amino acids differs depending on the substrate and that these differences are associated with the size, shape, and flexibility of the substrate and (2) the proximal residue Lys453 appears to play a critical role in the catalytic activity of P-450d, perhaps by participating in forming an intermolecular electron-transfer complex.     

Human salivary alpha-amylase Trp58 situated at subsite -2 is critical for enzyme activity.

The nonreducing end of the substrate-binding site of human salivary alpha-amylase contains two residues Trp58 and Trp59, which belong to beta2-alpha2 loop of the catalytic (beta/alpha)(8) barrel. While Trp59 stacks onto the substrate, the exact role of Trp58 is unknown. To investigate its role in enzyme activity the residue Trp58 was mutated to Ala, Leu or Tyr. Kinetic analysis of the wild-type and mutant enzymes was carried out with starch and oligosaccharides as substrates. All three mutants exhibited a reduction in specific activity (150-180-fold lower than the wild type) with starch as substrate. With oligosaccharides as substrates, a reduction in k(cat), an increase in K(m) and distinct differences in the cleavage pattern were observed for the mutants W58A and W58L compared with the wild type. Glucose was the smallest product generated by these two mutants in the hydrolysis oligosaccharides; in contrast, wild-type enzyme generated maltose as the smallest product. The production of glucose by W58L was confirmed from both reducing and nonreducing ends of CNP-labeled oligosaccharide substrates. The mutant W58L exhibited lower binding affinity at subsites -2, -3 and +2 and showed an increase in transglycosylation activity compared with the wild type. The lowered affinity at subsites -2 and -3 due to the mutation was also inferred from the electron density at these subsites in the structure of W58A in complex with acarbose-derived pseudooligosaccharide. Collectively, these results suggest that the residue Trp58 plays a critical role in substrate binding and hydrolytic activity of human salivary alpha-amylase.     

Characterization of a mannose-6-phosphate isomerase from Thermus thermophilus and increased L-ribose production by its R142N mutant

An uncharacterized gene from Thermus thermophilus, thought to encode a mannose-6-phosphate isomerase, was cloned and expressed in Escherichia coli. The maximal activity of the recombinant enzyme for L-ribulose isomerization was observed at pH 7.0 and 75°C in the presence of 0.5 mM Cu(2+). Among all of the pentoses and hexoses evaluated, the enzyme exhibited the highest activity for the conversion of L-ribulose to L-ribose, a potential starting material for many L-nucleoside-based pharmaceutical compounds. The active-site residues, predicted according to a homology-based model, were separately replaced with Ala. The residue at position 142 was correlated with an increase in L-ribulose isomerization activity. The R142N mutant showed the highest activity among mutants modified with Ala, Glu, Tyr, Lys, Asn, or Gln. The specific activity and catalytic efficiency (k(cat)/K(m)) for L-ribulose using the R142N mutant were 1.4- and 1.6-fold higher than those of the wild-type enzyme, respectively. The k(cat)/K(m) of the R142N mutant was 3.8-fold higher than that of Geobacillus thermodenitrificans mannose-6-phosphate isomerase, which exhibited the highest activity to date for the previously reported k(cat)/K(m). The R142N mutant enzyme produced 213 g/liter L-ribose from 300 g/liter L-ribulose for 2 h, with a volumetric productivity of 107 g liter(-1) h(-1), which was 1.5-fold higher than that of the wild-type enzyme.     

Amino acid residues in ribonuclease MC1 from bitter gourd seeds which are essential for uridine specificity

The ribonuclease MC1 (RNase MC1), isolated from seeds of bitter gourd (Momordica charantia), consists of 190 amino acids and is characterized by specific cleavage at the 5'-side of uridine. Site-directed mutagenesis was used to evaluate the contribution of four amino acids, Asn71, Val72, Leu73, and Arg74, at the alpha4-alpha5 loop between alpha4 and alpha5 helices for recognition of uracil base by RNase MC1. Four mutants, N71T, V72L, L73A, and R74S, in which Asn71, Val72, Leu73, and Arg74 in RNase MC1 were substituted for the corresponding amino acids, Thr, Leu, Ala, and Ser, respectively, in a guanylic acid preferential RNase NW from Nicotiana glutinosa, were prepared and characterized with respect to enzymatic activity. Kinetic analysis with a dinucleoside monophosphate, CpU, showed that the mutant N71T exhibited 7.0-fold increased K(m) and 2.3-fold decreased k(cat), while the mutant L73A had 14.4-fold increased K(m), although it did retain the k(cat) value comparable to that of the wild-type. In contrast, replacements of Val72 and Arg74 by the corresponding amino acids Leu and Ser, respectively, had little effect on the enzymatic activity. This observation is consistent with findings in the crystal structure analysis that Asn71 and Leu73 are responsible for a uridine specificity for RNase MC1. The role of Asn71 in enzymatic reaction of RNase MC1 was further investigated by substituting amino acids Ala, Ser, Gln, and Asp. Our observations suggest that Asn71 has at least two roles: one is base recognition by hydrogen bonding, and the other is to stabilize the conformation of the alpha4-alpha5 loop by hydrogen bonding to the peptide backbone, events which possibly result in an appropriate orientation of the alpha-helix (alpha5) containing active site residues. Mutants N71T and N71S showed a remarkable shift from uracil to guanine specificity, as evaluated by cleavage of CpG, although they did exhibit uridine specificity against yeast RNA and homopolynucleotides.     

Substrate specificity of the Escherichia coli outer membrane protease OmpP

Escherichia coli OmpP is an F episome-encoded outer membrane protease that exhibits 71% amino acid sequence identity with OmpT. These two enzymes cleave substrate polypeptides primarily between pairs of basic amino acids. We found that, like OmpT, purified OmpP is active only in the presence of lipopolysaccharide. With optimal peptide substrates, OmpP exhibits high catalytic efficiency (k(cat)/K(m) = 3.0 x 10(6) M(-1)s(-1)). Analysis of the extended amino acid specificity of OmpP by substrate phage revealed that both Arg and Lys are strongly preferred at the P1 and P1' sites of the enzyme. In addition, Thr, Arg, or Ala is preferred at P2; Leu, Ala, or Glu is preferred at P4; and Arg is preferred at P3'. Notable differences in OmpP and OmpT specificities include the greater ability of OmpP to accept Lys at the P1 or P1', site as well as the prominence of Ser at P3 in OmpP substrates. Likewise, the OmpP P1 site could better accommodate Ser; as a result, OmpP was able to cleave a peptide substrate between Ser-Arg about 120 times more efficiently than was OmpT. Interestingly, OmpP and OmpT cleave peptides with three consecutive Arg residues at different sites, a difference in specificity that might be important in the inactivation of cationic antimicrobial peptides. Accordingly, we show that the presence of an F' episome results in increased resistance to the antimicrobial peptide protamine both in ompT mutants and in wild-type E. coli cells.     

Kinetic studies on drug-resistant variants of Escherichia coli thymidylate synthase: functional effects of amino acid substitutions at residue 4.

A naturally occurring mutant of human thymidylate synthase (hTS) that contains a Tyr to His mutation at residue 33 was found to confer 4-fold resistance to 5-fluoro-2'-deoxyuridine (FdUrd), a prodrug of 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP). The crystal structure of hTS implicated this Tyr residue in a drug resistance mechanistic role that may include both substrate binding and catalysis (Schiffer et al., Biochemistry, 34, 16279-16287, 1995). Because of the existence of a defined kinetic scheme and the development of a bacterial expression vector for the overproduction of Escherichia coli TS (ecTS), we chose to initially study the corresponding residue in the bacterial enzyme, Tyr 4 of ecTS. Nine mutant ecTS enzymes that differed in sequence at position 4 were generated. Mutants with a charged or polar side chain (Ser, Cys, Asp, and Arg) and Gly precipitated in the cell paste, resulting in no catalytic activity in cell-free extracts. Although most of the His 4 mutant precipitated, sufficient amounts remained in the cell-free extract to permit isolation to near homogeneity. Wild-type ecTS and mutants with a hydrophobic side chain (Phe, Ile, and Val) were expressed at nearly 30% of the total cellular protein. The k(cat) values for the isolatable mutants were 2- to 10-fold lower than that of the wild-type enzyme, while the K(m) values for 2'-deoxyuridylate (dUMP) and 5,10-methylenetetrahydrofolate (CH(2)H(4)folate) were similar for all the mutants. Dissociation constants for binary complex formation determined by stopped-flow spectroscopy were similar for the wild-type and mutant enzymes for both dUMP and 2'-deoxythymidylate, indicating that this mutation does not significantly alter the binding of the natural nucleotide ligands. However, each mutant enzyme had three- to 5-fold lower affinity for FdUMP in the binary complex compared with the wild-type enzyme, and only His 4 showed a lower affinity for FdUMP in the ternary complex. Analysis of k(burst) showed that the initial binding of CH(2)H(4)folate is weaker for each mutant compared to the wild-type enzyme and that lower k(cat) values were due to compromised rates that govern the chemical transformation of bound substrates to bound products.     

Removal of distal protein-water hydrogen bonds in a plant epoxide hydrolase increases catalytic turnover but decreases thermostability

A putative proton wire in potato soluble epoxide hydrolase 1, StEH1, was identified and investigated by means of site-directed mutagenesis, steady-state kinetic measurements, temperature inactivation studies, and X-ray crystallography. The chain of hydrogen bonds includes five water molecules coordinated through backbone carbonyl oxygens of Pro(186), Leu(266), His(269), and the His(153) imidazole. The hydroxyl of Tyr(149) is also an integrated component of the chain, which leads to the hydroxyl of Tyr(154). Available data suggest that Tyr(154) functions as a final proton donor to the anionic alkylenzyme intermediate formed during catalysis. To investigate the role of the putative proton wire, mutants Y149F, H153F, and Y149F/H153F were constructed and purified. The structure of the Y149F mutant was solved by molecular replacement and refined to 2.0 A resolution. Comparison with the structure of wild-type StEH1 revealed only subtle structural differences. The hydroxyl group lost as a result of the mutation was replaced by a water molecule, thus maintaining a functioning hydrogen bond network in the proton wire. All mutants showed decreased catalytic efficiencies with the R,R-enantiomer of trans-stilbene oxide, whereas with the S,S-enantiomer, k (cat)/K (M) was similar or slightly increased compared with the wild-type reactions. k (cat) for the Y149F mutant with either TSO enantiomer was increased; thus the lowered enzyme efficiencies were due to increases in K (M). Thermal inactivation studies revealed that the mutated enzymes were more sensitive to elevated temperatures than the wild-type enzyme. Hence, structural alterations affecting the hydrogen bond chain caused increases in k (cat) but lowered thermostability.     

Protease C of Erwinia chrysanthemi: the crystal structure and role of amino acids Y228 and E189.

PrtC, a metallo-protease secreted by Erwinia chrysanthemi, is a member of the serralysin family and hence belongs to the metzincin superfamily. While the crystal structures of representatives of all metzincin subfamilies have been elucidated in the past, there is still some controversy about the reaction mechanism and the role of certain characteristic amino acids in the active centre. In this study, we probed the role of Tyr228 and Glu189 by site-directed mutagenesis and X-ray crystallography. There is evidence that these residues participate in catalysis, although conflicting hypotheses have been proposed. The crystal structures of wild-type and mutants have been refined to an R(free) of about 0.20 at resolutions of 2.0 A or better. Exchange of Glu189 versus alanine reduces the catalytic efficiency to less than 0.5 % using resorufin casein as substrate and to about 3 % using an assay utilising the thiol ester Ac-Pro-Leu-Gly-[(S)Leu]-Leu-Gly-OEt. The drop in activity is caused by a reduction in k(cat) while the K(M) values are virtually the same. In the resorufin casein assay, the mutant Y228F shows about 3 % of the wild-type activity and in the thiol ester assay this increases to about 56 %. In the latter case, the K(M) value of the mutant is increased from 5.3 mM to 9.0 mM with only little reduction in k(cat). The different behaviour of this mutant with respect to the two substrates can be explained by a switch in the rate-determining step during catalysis. The study presented here provides clear evidence that Glu189 of the HEXXHXXGXXH motif is the catalytic base, while Tyr228 is more likely involved in substrate binding and the stabilisation of the tetrahedral transition state.     

Role of the electrostatic loop charged residues in Cu,Zn superoxide dismutase.

We have expressed and characterized a mutant of Xenopus laevis Cu,Zn superoxide dismutase in which four highly conserved charged residues belonging to the electrostatic loop have been replaced by neutral side chains: Lys120 --> Leu, Asp130 --> Gln, Glu131 --> Gln, and Lys134 --> Thr. At low ionic strength, the mutant enzyme is one of the fastest superoxide dismutases ever assayed (k = 6.7 x 10(9) M(-1) s(-1), at pH 7 and mu = 0.02 M). Brownian dynamics simulations give rise to identical enzyme-substrate association rates for both wild-type and mutant enzymes, ruling out the possibility that enhancement of the activity is due to pure electrostatic factors. Comparative analysis of the experimental catalytic rate of the quadruple and single mutants reveals the nonadditivity of the mutation effects, indicating that the hyperefficiency of the mutant is due to a decrease of the energy barrier and/or to an alternative pathway for the diffusion of superoxide within the active site channel. At physiological ionic strength the catalytic rate of the mutant at neutral pH is similar to that of the wild-type enzyme as it is to the catalytic rate pH dependence. Moreover, mutation effects are additive. These results show that, at physiological salt conditions, electrostatic loop charged residues do not influence the diffusion pathway of the substrate and, if concomitantly neutralized, are not essential for high catalytic efficiency of the enzyme, pointing out the role of the metal cluster and of the invariant Arg141 in determining the local electrostatic forces facilitating the diffusion of the substrate towards the active site.     

Substrate binding mode and its implication on drug design for botulinum neurotoxin A

The seven antigenically distinct serotypes of Clostridium botulinum neurotoxins, the causative agents of botulism, block the neurotransmitter release by specifically cleaving one of the three SNARE proteins and induce flaccid paralysis. The Centers for Disease Control and Prevention (CDC) has declared them as Category A biowarfare agents. The most potent among them, botulinum neurotoxin type A (BoNT/A), cleaves its substrate synaptosome-associated protein of 25 kDa (SNAP-25). An efficient drug for botulism can be developed only with the knowledge of interactions between the substrate and enzyme at the active site. Here, we report the crystal structures of the catalytic domain of BoNT/A with its uncleavable SNAP-25 peptide (197)QRATKM(202) and its variant (197)RRATKM(202) to 1.5 A and 1.6 A, respectively. This is the first time the structure of an uncleavable substrate bound to an active botulinum neurotoxin is reported and it has helped in unequivocally defining S1 to S5' sites. These substrate peptides make interactions with the enzyme predominantly by the residues from 160, 200, 250 and 370 loops. Most notably, the amino nitrogen and carbonyl oxygen of P1 residue (Gln197) chelate the zinc ion and replace the nucleophilic water. The P1'-Arg198, occupies the S1' site formed by Arg363, Thr220, Asp370, Thr215, Ile161, Phe163 and Phe194. The S2' subsite is formed by Arg363, Asn368 and Asp370, while S3' subsite is formed by Tyr251, Leu256, Val258, Tyr366, Phe369 and Asn388. P4'-Lys201 makes hydrogen bond with Gln162. P5'-Met202 binds in the hydrophobic pocket formed by the residues from the 250 and 200 loop. Knowledge of interactions between the enzyme and substrate peptide from these complex structures should form the basis for design of potent inhibitors for this neurotoxin.     

Substrate specificity determinants of human macrophage elastase (MMP-12) based on the 1.1 A crystal structure

The macrophage elastase enzyme (MMP-12) expressed mainly in alveolar macrophages has been identified in the mouse lung as the main destructive agent associated with cigarette smoking, which gives rise to emphysema, both directly via elastin degradation and indirectly by disturbing the proteinase/antiproteinase balance via inactivation of the alpha1-proteinase inhibitor (alpha1-PI), the antagonist of the leukocyte elastase. The catalytic domain of human recombinant MMP-12 has been crystallized in complex with the broad-specificity inhibitor batimastat (BB-94). The crystal structure analysis of this complex, determined using X-ray data to 1.1 A and refined to an R-value of 0.165, reveals an overall fold similar to that of other MMPs. However, the S-shaped double loop connecting strands III and IV is fixed closer to the beta-sheet and projects its His172 side-chain further into the rather hydrophobic active-site cleft, defining the S3 and the S1-pockets and separating them from each other to a larger extent than is observed in other MMPs. The S2-site is planar, while the characteristic S1'-subsite is a continuous tube rather than a pocket, in which the MMP-12-specific Thr215 replaces a Val residue otherwise highly conserved in almost all other MMPs. This alteration might allow MMP-12 to accept P1' Arg residues, making it unique among MMPs. The active-site cleft of MMP-12 is well equipped to bind and efficiently cleave the AlaMetPhe-LeuGluAla sequence in the reactive-site loop of alpha1-PI, as occurs experimentally. Similarities in contouring and particularly a common surface hydrophobicity both inside and distant from the active-site cleft explain why MMP-12 shares many substrates with matrilysin (MMP-7). The MMP-12 structure is an excellent template for the structure-based design of specific inhibitors for emphysema therapy and for the construction of mutants to clarify the role of this MMP.     

Function of the fully conserved residues Asp99, Tyr52 and Tyr73 in phospholipase A2

In the active centre of pancreatic phospholipase A2 His48 is at hydrogen-bonding distance to Asp99. This Asp-His couple is assumed to act together with a water molecule as a catalytic triad. Asp99 is also linked via an extended hydrogen bonding system to the side chains of Tyr52 and Tyr73. To probe the function of the fully conserved Asp99, Tyr52 and Tyr73 residues in phospholipase A2, the Asp99 residue was replaced by Asn, and each of the two tyrosines was separately replaced by either a Phe or a Gln. The catalytic and binding properties of the Phe52 and Phe73 mutants did not change significantly relative to the wild-type enzyme. This rules out the possibility that either one of the two Tyr residues in the wild-type enzyme can function as an acyl acceptor or proton donor in catalysis. The Gln73 mutant could not be obtained in any significant amounts probably due to incorrect folding. The Gln52 mutant was isolated in low yield. This mutant showed a large decrease in catalytic activity while its substrate binding was nearly unchanged. The results suggest a structural role rather than a catalytic function of Tyr52 and Tyr73. Substitution of asparagine for aspartate hardly affects the binding constants for both monomeric and micellar substrate analogues. Kinetic characterization revealed that the Asn99 mutant has retained no less than 65% of its enzymatic activity on the monomeric substrate rac 1,2-dihexanoyldithio-propyl-3-phosphocholine, probably due to the fact that during hydrolysis of monomeric substrate by phospholipase A2 proton transfer is not the rate-limiting step.(ABSTRACT TRUNCATED AT 250 WORDS)     

Catalysis by Escherichia coli ribonuclease HI is facilitated by a phosphate group of the substrate

To investigate the role of the phosphate group 3' to the scissile phosphodiester bond of the substrate in the catalytic mechanism of Escherichia coli ribonuclease HI (RNase HI), we have used modified RNA-DNA hybrid substrates carrying a phosphorothioate substitution at this position or lacking this phosphate group for the cleavage reaction. Kinetic parameters of the H124A mutant enzyme, in which His(124) was substituted with Ala, as well as those of the wild-type RNase HI, were determined. Substitution of the pro-R(p)-oxygen of the phosphate group 3' to the scissile phosphodiester bond of the substrate with sulfur reduced the k(cat) value of the wild-type RNase HI by 6.9-fold and that of the H124A mutant enzyme by only 1. 9-fold. In contrast, substitution of the pro-S(p)-oxygen of the phosphate group at this position with sulfur had little effect on the k(cat) value of the wild-type and H124A mutant enzymes. The results obtained for the substrate lacking this phosphate group were consistent with those obtained for the substrates with the phosphorothioate substitutions. In addition, a severalfold increase in the K(m) value was observed by the substitution of the pro-R(p)-oxygen of the substrate with sulfur or by the substitution of His(124) of the enzyme with Ala, suggesting that a hydrogen bond is formed between the pro-R(p)-oxygen and His(124). These results allow us to propose that the pro-R(p)-oxygen contributes to orient His(124) to the best position for the catalytic function through the formation of a hydrogen bond.     

The choline binding site of phospholipase C (Bacillus cereus): insights into substrate specificity

The phosphatidylcholine-preferring phospholipase C from Bacillus cereus (PLC(Bc)) is a 28.5 kDa enzyme with three zinc ions in its active site. The roles that a number of amino acid residues play as zinc ligands and in binding and catalysis have been elucidated. Recent mechanistic studies indicate that the rate of the reaction is limited by a proton-transfer step during chemical hydrolysis and not substrate binding or product release. An X-ray structure of PLC(Bc) complexed with a phosphonate inhibitor related to phosphatidylcholine revealed that the three amino acid residues Glu4, Tyr56, and Phe66 comprise the choline binding pocket. However, because the contributions that these three residues make to substrate recognition and specificity were unknown, a series of site-specific mutants for Glu4, Tyr56, and Phe66 were constructed by PCR mutagenesis. On the basis of a comparison of their respective CD spectra and melting temperatures, it appears that the mutants adopt folded structures in solution that are virtually identical to that of wild-type PLC(Bc). The kinetic parameters k(cat) and K(m) for the hydrolysis of the three soluble substrates 1, 2-dihexanoyl-sn-glycero-3-phosphocholine (C6PC), 1, 2-dihexanoyl-sn-glycero-3-phosphoethanolamine (C6PE), and 1, 2-dihexanoyl-sn-glycero-3-phospho-L-serine (C6PS) at concentrations below their corresponding critical micelle concentration (cmc) values were determined for each mutant. Replacement of Phe66 with a nonaromatic residue dramatically decreased k(cat) (approximately 200-fold) and reduced PLC(Bc) activity toward C6PC, C6PE, and C6PS, whereas changes to Glu4 and Tyr56 typically led to much more modest losses in catalytic efficiencies. Mutations of Glu4 had relatively little effect upon k(cat) and K(m) for C6PS, but they significantly influenced K(m) for C6PC and C6PE. Replacing Tyr56 with nonaromatic residues also affects catalytic efficiency, albeit to a much lesser degree than the corresponding changes at position 66. However, the presence of an aromatic residue at position 56 seems to confer some substrate selectivity for C6PC and C6PE, which bear a positive charge on the headgroup, relative to C6PS, which has no net charge on the headgroup; this increase in specificity arises largely from a reduced k(cat) for C6PS.