Connecting Active‐Site Loop Conformations and Catalysis in Triosephosphate Isomerase: Insights from a Rare Variation at Residue 96 in the Plasmodial Enzyme

Despite extensive research into triosephosphate isomerases (TIMs), there exists a gap in understanding of the remarkable conjunction between catalytic loop‐6 (residues 166–176) movement and the conformational flip of Glu165 (catalytic base) upon substrate binding that primes the active site for efficient catalysis. The overwhelming occurrence of serine at position 96 (98 % of the 6277 unique TIM sequences), spatially proximal to E165 and the loop‐6 residues, raises questions about its role in catalysis. Notably, Plasmodium falciparum TIM has an extremely rare residue—phenylalanine—at this position whereas, curiously, the mutant F96S was catalytically defective. We have obtained insights into the influence of residue 96 on the loop‐6 conformational flip and E165 positioning by combining kinetic and structural studies on the PfTIM F96 mutants F96Y, F96A, F96S/S73A, and F96S/L167V with sequence conservation analysis and comparative analysis of the available apo and holo structures of the enzyme from diverse organisms.     

Separation of the two reactions, oxidation and isomerization, catalyzed by Streptomyces cholesterol oxidase

Site-directed mutagenesis was used to identify key amino acid residues of the cholesterol oxidase from Streptomyces sp., which catalyzes the oxidation of cholesterol and the isomerization of 5-cholesten-3-one. Eight mutant enzymes were constructed and the following amino acid substitutions were identified: N318A, N318H, E356A, E356D, H441A, H441N, N480A and N480Q. Mutants N318A and N318H retained both oxidation and isomerization activities. The mutant E356D retained oxidation but not isomerization activity. On the other hand, mutants N480A and N480Q showed no oxidation activity but retained their isomerization activities. The two catalytic reactions, oxidation and isomerization, in cholesterol oxidase were thus successfully separated. When the H441A or H441N mutation was introduced, both the oxidase and isomerase activities were completely lost. The H441, E356 and N480 residues thus appear to participate in the catalysis of cholesterol oxidase, whereas N318 does not. An analysis of the products of these mutant enzymes suggested that the previously proposed 6-hydroxylation reaction by cholesterol oxidase is actually autooxidation from 5-cholesten-3-one. Kinetic studies of the purified wild-type and mutant enzymes showed that the k(cat)/Km values for oxidation in E356D and for isomerization in N480A increased six- and threefold, respectively, over those in the wild-type. These mutational effects and the reaction mechanisms are discussed in terms of the three-dimensional structure of the enzyme constructed on the basis of homology modeling.      

Use of a minimum perturbation approach to predict TIM mutant structures

A minimum perturbation conformational search approach is usedto model the structures of the yeast triosephosphate isomerase(TIM) single mutant in which the catalytic base Glul65 is changedto Asp, and the double mutant in which Glul65 is changed toAsp and Ser96 to Pro. In chicken TIM this double mutant is referredto as a pseudo–revertant because some of the catalyticactivity lost due to the first mutation is regained when thesecond mutation occurs. Three minimum energy structures werecalculated for the Asp 165 conformation in the yeast TEM singlemutant and another three for the double mutant One of the calculatedminimum energy conformations for Aspl65 in the E165D structureagrees well with the X–ray structure. However, this conformationis not that of the lowest energy and is not one of the threemost common conformers for Asp found by Ponder and Richards.This suggests that when an amino acid is introduced it may notbe able to conform to the more general rules that apply to proteinstructures of evolutionary origin. While the van der Waals energylargely determines the allowed minima, the relative rankingof the final minima is determined by electrostatic effects andcan therefore be affected by the inclusion of crystal watersin the calculation. When the E165D calculation is repeated withan active–site water molecule fixed in its E165D X–raystructure position, the relative ranking of the minima shiftsand the X–ray conformation for Asp 165 is the lowest interactionenergy conformer. Two of the E165D calculated minimum energystructures are essentially identical to two of the S96P/E165Dminima. All of the calculated minima for both the E165D andS96P/E165D mutants position the Asp side chain such that theanti–orbital, and not the more basic syn–orbital,of the carboxylate would be utilized for proton abstraction.This observation may explain why the chicken TIM S96P/E165Dmutant, for which the X-ray structure indicates that the syn–orbitalis used, is a pseudo–revertant while the yeast TIM doublemutant is not; no X–ray structure is available for thelatter. The multiplicity of minima found in the present analysismakes clear that predicting the exact orientation of a singleside chain is not as simple as might be expected.     

A potential antitumor drug (arginine deiminase) reengineered for efficient operation under physiological conditions

Arginine deiminase (ADI, EC 3.5.3.6) is a potential antitumor drug for the treatment of arginine‐auxotrophic tumors such as hepatocellular carcinomas (HCCs) and melanomas, and studies on human lymphatic leukemia cell lines have confirmed that ADI has antiangiogenic activity. Recent studies showed that a combination of taxane and ADI‐PEG20, which induces caspase‐independent apoptosis, is more effective than taxane monotherapy for prostate cancer. The main limitation of ADI from Pseudomonas plecoglossicida (PpADI) and of many other ADI enzymes lies in their pH‐dependent activity profile. PpADI has a pH optimum at 6.5 and a pH shift from 6.5 to 7.5 results in an ～80 % activity drop (the pH of human plasma is 7.35 to 7.45). In 2010, we reported a proof of concept for ADI engineering by directed evolution that resulted in variant M2 (K5T/D44E/H404R). M2 has a pH optimum of pH 7.0, a fourfold higher k_cat value than the wild‐type PpADI (pH 7.4, 0.5 M phosphate buffer), and an increased K_m value for substrate arginine. In our latest work, variants M5 (K5T/D38H/D44E/A128T/H404R) and M6 (K5T/D38H/D44E/A128T/E296K/H404R) were generated by directed evolution by employing PBS buffer (pH 7.4), which mimics physiological conditions. The S_0.5 value of parent M3 (K5T/D44E/A128T/H404R) decreased from 2.01 to 1.48 mM (M5) and 0.81 mM (M6). The S_0.5 value of M6 (0.81 mM ) is lower than that of wild‐type PpADI (1.30 mM ); the k_cat values improved from 0.18 s^?1 (wild‐type PpADI) to 17.56 s^?1 (M5, 97.6‐fold) and 11.64 s^?1 (M6, 64.7‐fold).     

Protein Engineering of the Antitumor Enzyme PpADI for Improved Thermal Resistance

Arginine deiminase (ADI, EC 3.5.3.6) is a potential antitumor drug for the treatment of arginine‐auxotrophic tumors such as hepatocellular carcinomas (HCCs) and melanomas. Studies in human lymphatic leukemia cell lines have confirmed the anti‐angiogenic activity of ADI. Activity and thermal resistance limit the efficacy of ADI in treatment of auxotrophic tumors. Previously, we reengineered ADI from Pseudomonas plecoglossicida (PpADI) for improved activity under physiological conditions (37 °C, PBS buffer, pH 7.4) by two rounds of directed evolution and combination of beneficial substitutions through site‐directed mutagenesis. The best variant, PpADI M6 (K5T/D38H/D44E/A128T/E296K/H404R), showed a 64.7‐fold improvement in k_cat value and a 37.6 % decreased S_0.5 value under physiological conditions. However, M6 lost rapidly its activity (half‐life of ～2 days at 37 °C). Here we report the re‐engineering of PpADI M6 for improved thermal resistance by directed evolution in order to increase its half‐life under physiological conditions. Directed evolution and recombination of the two most beneficial positions yielded variant PpADI M9 (K5T/D38H/D44E/A128T/V140L/E296K/F325L/H404R), for which the T_m value increased from 47 (M6) to 54 °C (M9); this corresponds to an increased half‐life from ～2 days (M6) to ～3.5 days (M9) under physiological conditions. Structure analysis of the homology model of M9 showed that the beneficial substitutions V140L and F325L likely promote the formation of tetrameric PpADI, which has greater thermal resistance than dimeric PpADI.     

Cucurbitacins of Cucumis prophetarum and Cucumis prophetarum

Cucurbitacin B, isocucurbitacin B, dihydrocucurbitacin B, cucurbitacin E, dihydrocucurbitacin E, isocucurbitacin D, dihydroisocucurbitacin D, cucurbitacin I, dihydrocucurbitacin I, cucurbitacin Q1, and dihydrocucurbitacin Q1 were identified for the first time as constituents of Cucumis prophetarum L.. Cucurbitacin B, cucurbitacin O, cucurbitacin P, cucurbitacin Q1, dihydrocucurbitacin Q1, isocucurbitacin E, and dihydroisocucurbitacin E also were identified as constituents of C. prophetarum Jusl. ssp. Dissectus. Isocucurbitacin E and dihydroisocucurbitacin E were isolated for the first time in nature. The chemical structures were determined with extensive spectroscopic analysis including 2D NMR, ¹H NMR, ¹³C NMR, correlated spectroscopy (COSY), heteronuclear chemical shift correlation (HETCOR), attached proton test (APT), and distortionless enhancement by polarization transfer (DEPT).     

Structural Analysis Reveals the Substrate‐Binding Mechanism for the Expanded Substrate Specificity of Mutant meso‐Diaminopimelate Dehydrogenase

A meso‐diaminopimelate dehydrogenase (DAPDH) from Clostridium tetani E88 (CtDAPDH) was found to have low activity toward the D ‐amino acids other than its native substrate. Site‐directed mutagenesis similar to that carried out on the residues mutated by Vedha‐Peters et al. resulted in a mutant enzyme with highly improved catalytic ability for the synthesis of D ‐amino acids. The crystal structures of the CtDAPDH mutant in apo form and in complex with meso‐diaminopimelate (meso‐DAP), D ‐leucine (D ‐leu), and 4‐methyl‐2‐oxopentanoic acid (MOPA) were solved. meso‐DAP was found in an area outside the catalytic cavity; this suggested a possible two‐step substrate‐binding mechanism for meso‐DAP. D ‐leu and MOPA each bound both to Leu154 and to Gly155 in the open form of CtDAPDH, and structural analysis revealed the molecular basis for the expanded substrate specificity of the mutant meso‐diaminopimelate dehydrogenases.     

Structural Insights into the Recovery of Aldolase Activity in N‐Acetylneuraminic Acid Lyase by Replacement of the Catalytically Active Lysine with γ‐Thialysine by Using a Chemical Mutagenesis Strategy

Chemical modification has been used to introduce the unnatural amino acid γ‐thialysine in place of the catalytically important Lys165 in the enzyme N‐acetylneuraminic acid lyase (NAL). The Staphylococcus aureus nanA gene, encoding NAL, was cloned and expressed in E. coli. The protein, purified in high yield, has all the properties expected of a class I NAL. The S. aureus NAL which contains no natural cysteine residues was subjected to site‐directed mutagenesis to introduce a cysteine in place of Lys165 in the enzyme active site. Subsequently chemical mutagenesis completely converted the cysteine into γ‐thialysine through dehydroalanine (Dha) as demonstrated by ESI‐MS. Initial kinetic characterisation showed that the protein containing γ‐thialysine regained 17 % of the wild‐type activity. To understand the reason for this lower activity, we solved X‐ray crystal structures of the wild‐type S. aureus NAL, both in the absence of, and in complex with, pyruvate. We also report the structures of the K165C variant, and the K165‐γ‐thialysine enzyme in the presence, or absence, of pyruvate. These structures reveal that γ‐thialysine in NAL is an excellent structural mimic of lysine. Measurement of the pH‐activity profile of the thialysine modified enzyme revealed that its pH optimum is shifted from 7.4 to 6.8. At its optimum pH, the thialysine‐containing enzyme showed almost 30 % of the activity of the wild‐type enzyme at its pH optimum. The lowered activity and altered pH profile of the unnatural amino acid‐containing enzyme can be rationalised by imbalances of the ionisation states of residues within the active site when the pK_a of the residue at position 165 is perturbed by replacement with γ‐thialysine. The results reveal the utility of chemical mutagenesis for the modification of enzyme active sites and the exquisite sensitivity of catalysis to the local structural and electrostatic environment in NAL.     

Effects of mutations in the calcium-binding sites of recoverin on its calcium affinity: evidence for successive filling of the calcium binding sites

A molecule of the photoreceptor Ca²⁺-binding protein recoverincontains four potential EF-hand Ca²⁺-binding sites, of whichonly two, the second and the third, are capable of binding calciumions. We have studied the effects of substitutions in the second,third and fourth EF-hand sites of recoverin on its Ca²⁺-bindingproperties and some other characteristics, using intrinsic fluorescence,circular dichroism spectroscopy and differential scanning microcalorimetry.The interaction of the two operating binding sites of wild-typerecoverin with calcium increases the protein's thermal stability,but makes the environment around the tryptophan residues moreflexible. The amino acid substitution in the EF-hand 3 (E121Q)totally abolishes the high calcium affinity of recoverin, whilethe mutation in the EF-hand 2 (E85Q) causes only a moderatedecrease in calcium binding. Based on this evidence, we suggestthat the binding of calcium ions to recoverin is a sequentialprocess with the EF-hand 3 being filled first. Estimation ofCa²⁺-binding constants according to the sequential binding schemegave the values 3.7 x 10⁶ and 3.1 x 10⁵ M^–1 for thirdand second EF-hands, respectively. The substitutions in theEF-hand 2 or 3 (or in both the sites simultaneously) do notdisturb significantly either tertiary or secondary structureof the apo-protein. Amino acid substitutions, which have beendesigned to restore the calcium affinity of the EF-hand 4 (G160D,K161E, K162N, D165G and K166Q), increase the calcium capacityand affinity of recoverin but also perturb the protein structureand decrease the thermostability of its apo-form.     

Multiple sequence alignment-inspired mutagenesis of marine epoxide hydrolase of Mugil cephalus for enhancing enantioselective hydrolytic activity

The marine fish microsomal epoxide hydrolase (mEH) of Mugil cephalus was engineered to enhance the enantioselective hydrolytic activity by multiple sequence alignment-inspired mutagenesis. The amino acid sequences of Aspergillus niger, Rhodotorula glutinis, zebra fish and human mEH were aligned and analyzed for identifying target amino acids. Single-point mutants (Q170K, E186K, E378D) and double-point mutants (E378D-Q170K, E378D-Y348F, E378D-Y348H) were developed and their hydrolytic activities were compared. The double-point mutant, E378D-Q170K, exhibited an enhanced hydrolytic activity by 4.6-fold, compared to the wild-type M. cephalus mEH. Enantiopure (S)-styrene oxide could be readily prepared with high enantiopurity more than 99%ee by using the double-point mutant.     

Effects of a buried cysteine-to-serine mutation on yeast triosephosphate isomerase structure and stability

All the members of the triosephosphate isomerase (TIM) family possess a cystein residue (Cys126) located near the catalytically essential Glu165. The evolutionarily conserved Cys126, however, does not seem to play a significant role in the catalytic activity. On the other hand, substitution of this residue by other amino acid residues destabilizes the dimeric enzyme, especially when Cys is replaced by Ser. In trying to assess the origin of this destabilization we have determined the crystal structure of Saccharomyces cerevisiae TIM (ScTIM) at 1.86 Å resolution in the presence of PGA, which is only bound to one subunit. Comparisons of the wild type and mutant structures reveal that a change in the orientation of the Ser hydroxyl group, with respect to the Cys sulfhydryl group, leads to penetration of water molecules and apparent destabilization of residues 132–138. The latter results were confirmed by means of Molecular Dynamics, which showed that this region, in the mutated enzyme, collapses at about 70 ns.     

Development of a Novel Biocatalyst for the Resolution of rac‐Pantolactone

A novel L ‐pantolactone hydrolase, Lph, from Agrobacterium tumefaciens Lu681 was characterized, which stereospecifically hydrolyses L ‐pantolactone to L ‐pantoic acid yielding D ‐pantolactone with > 95% enantiomeric excess. The enzyme was found to be a 30 kDa‐Zn²⁺‐hydrolase with a K_m for L ‐pantolactone of 7 mM and a V_max of 30 U/mg. The corresponding lph gene was identified as an 807 bp ORF and cloned into E. coli. It was overexpressed under control of P_tac and P_rha yielding enzyme activities of up to 600 U/g dry weight. Resolution of d,l ‐pantolactone in repeated batches with isolated Lph and enzyme recovery by membrane filtration gave D ‐pantolactone with 50% yield and 90–95% ee over 6 days. Covalent immobilization to EupergitC led to a stable biocatalyst easy to handle in a repeated batch production of D ‐pantolactone. Further improvements in the activity of Lph were achieved by directed evolution of the enzyme. Activities of mutants F62S, K197D and F100L were increased 2.3, 1.7, and 1.5 fold, respectively.     

Investigation of Structural Determinants for the Substrate Specificity in the Zinc‐Dependent Alcohol Dehydrogenase CPCR2 from Candida parapsilosis

Zinc‐dependent alcohol dehydrogenases (ADHs) are a class of enzymes applied in different biocatalytic processes ranging from lab to industrial scale. However, one drawback is the limited substrate range, necessitating a whole array of different ADHs for the relevant substrate classes. In this study, we investigated structural determinants of the substrate spectrum in the zinc‐dependent ADH carbonyl reductase 2 from Candida parapsilosis (CPCR2), combining methods of mutational analysis with in silico substrate docking. Assigned active site residues were genetically randomized, and the resulting mutant libraries were screened with a selection of challenging carbonyl substrates. Three variants (C57A, W116K, and L119M) with improved activities toward different substrates were detected at neighboring positions in the active site. Thus, all possible combinations of the mutations were generated and characterized for their substrate specificity, yielding several improved variants. The most interesting were a C57A variant, with a 27‐fold increase in specific activity for 4′‐acetamidoacetophenone, and the double mutant CPCR2 B16‐(C57A, L119M), with a 45‐fold improvement in the k_cat?K_M^?1 value. The obtained variants were further investigated by in silico docking experiments. The results indicate that the mentioned residues are structural determinants of the substrate specificity of CPCR2, being major players in the definition of the active site. Comparison of these results with closely related enzymes suggests that these might even be transferred to other ADHs.     

Evaluating p97 Inhibitor Analogues for Their Domain Selectivity and Potency against the p97–p47 Complex

We previously found that p97 ATPase inhibitors 2‐(2‐amino‐1H‐benzo[d]imidazol‐1‐yl)‐N‐benzyl‐8‐methoxyquinazolin‐4‐amine ( ML240 ) and 2‐(2H‐benzo[b][1,4]oxazin‐4(3H)‐yl)‐N‐benzyl‐5,6,7,8‐tetrahydroquinazolin‐4‐amine ( ML241 ) specifically target the D2 domain of wild‐type p97. In addition, one of the major p97 cofactors, p47, decreases their potencies by ～50‐fold. In contrast, N²,N⁴‐dibenzylquinazoline‐2,4‐diamine ( DBeQ ) targets both the D1 and D2 domains and shows only a four‐ to sixfold decrease in potency against the p97–p47 complex. To elucidate structure–activity relationships for the inhibitors, we screened 200 p97 inhibitor analogues for their ability to inhibit the ATPase activity of either or both of the D1 or D2 domains, as well for their effects on p47 potency. The selectivity of 29 of these compounds was further examined by eight‐dose titrations. Four compounds showed modest selectivity for inhibiting the ATPase activity of D1. Eleven compounds inhibited D2 with greater potencies, and four showed similar potencies against D1 and D2. p47 decreased the potencies of the majority of the compounds and increased the potencies of five compounds. These results highlight the possibility of developing domain‐selective and complex‐specific p97 inhibitors in order to further elucidate the physiological roles of p97 and its cofactors.     

Alteration of the Diastereoselectivity of 3‐Methylaspartate Ammonia Lyase by Using Structure‐Based Mutagenesis

3‐Methylaspartate ammonia‐lyase (MAL) catalyzes the reversible amination of mesaconate to give both (2S,3S)‐3‐methylaspartic acid and (2S,3R)‐3‐methylaspartic acid as products. The deamination mechanism of MAL is likely to involve general base catalysis, in which a catalytic base abstracts the C3 proton of the respective stereoisomer to generate an enolate anion intermediate that is stabilized by coordination to the essential active‐site Mg^II ion. The crystal structure of MAL in complex with (2S,3S)‐3‐methylaspartic acid suggests that Lys331 is the only candidate in the vicinity that can function as a general base catalyst. The structure of the complex further suggests that two other residues, His194 and Gln329, are responsible for binding the C4 carboxylate group of (2S,3S)‐3‐methylaspartic acid, and hence are likely candidates to assist the Mg^II ion in stabilizing the enolate anion intermediate. In this study, the importance of Lys331, His194, and Gln329 for the activity and stereoselectivity of MAL was investigated by site‐directed mutagenesis. His194 and Gln329 were replaced with either an alanine or arginine, whereas Lys331 was mutated to a glycine, alanine, glutamine, arginine, or histidine. The properties of the mutant proteins were investigated by circular dichroism (CD) spectroscopy, kinetic analysis, and ¹H NMR spectroscopy. The CD spectra of all mutants were comparable to that of wild‐type MAL, and this indicates that these mutations did not result in any major conformational changes. Kinetic studies demonstrated that the mutations have a profound effect on the values of k_cat and k_cat/K_M; this implicates Lys331, His194 and Gln329 as mechanistically important. The ¹H NMR spectra of the amination and deamination reactions catalyzed by the mutant enzymes K331A, H194A, and Q329A showed that these mutants have strongly enhanced diastereoselectivities. In the amination direction, they catalyze the conversion of mesaconate to yield only (2S,3S)‐3‐methylaspartic acid, with no detectable formation of (2S,3R)‐3‐methylaspartic acid. The results are discussed in terms of a mechanism in which Lys331, His194, and Gln329 are involved in positioning the substrate and in formation and stabilization of the enolate anion intermediate.     

Folding and Unfolding of Two Mixed α/β Peptides

We present a molecular dynamics simulation study of two peptides containing α‐ and β‐amino acid residues. According to experiment, the two peptides differ in the dominant fold when solvated in methanol: one shows a helical fold, the other a β hairpin. The simulations at 300 and 340 K were done by starting from a NMR spectroscopic model structure and from an extended (denatured) structure. The typical structural features of the two peptides are reproduced and a folding/unfolding equilibrium is observed on the nanosecond timescale at 300 K. Analysis of proton–proton NOE distance bounds and backbone ³J coupling constants gives results consistent with the experimental data. We conclude that our simulations are complementary to the experiments by providing detailed information on the conformational distributions.     

Mutations Q93H and E97K in TPM2 Disrupt Ca-Dependent Regulation of Actin Filaments

Tropomyosin is a two-chain coiled coil protein, which together with the troponin complex controls interactions of actin with myosin in a Ca²⁺-dependent manner. In fast skeletal muscle, the contractile actin filaments are regulated by tropomyosin isoforms Tpm1.1 and Tpm2.2, which form homo- and heterodimers. Mutations in the TPM2 gene encoding isoform Tpm2.2 are linked to distal arthrogryposis and congenital myopathy—skeletal muscle diseases characterized by hyper- and hypocontractile phenotypes, respectively. In this work, in vitro functional assays were used to elucidate the molecular mechanisms of mutations Q93H and E97K in TPM2. Both mutations tended to decrease actin affinity of homo-and heterodimers in the absence and presence of troponin and Ca²⁺, although the effect of Q93H was stronger. Changes in susceptibility of tropomyosin to trypsin digestion suggested that the mutations diversified dynamics of tropomyosin homo- and heterodimers on the filament. The presence of Q93H in homo- and heterodimers strongly decreased activation of the actomyosin ATPase and reduced sensitivity of the thin filament to [Ca²⁺]. In contrast, the presence of E97K caused hyperactivation of the ATPase and increased sensitivity to [Ca²⁺]. In conclusion, the hypo- and hypercontractile phenotypes associated with mutations Q93H and E97K in Tpm2.2 are caused by defects in Ca²⁺-dependent regulation of actin–myosin interactions.     

Engineered dimer interface mutants of triosephosphate isomerase: the role of inter-subunit interactions in enzyme function and stability

The role of inter-subunit interactions in maintaining optimal catalytic activity in triosephosphate isomerase (TIM) has been probed, using the Plasmodium falciparum enzyme as a model. Examination of subunit interface contacts in the crystal structures suggests that residue 75 (Thr, conserved) and residue 13 (Cys, variable) make the largest number of inter-subunit contacts. The mutants Cys13Asp (C13D) and Cys13Glu (C13E) have been constructed and display significant reduction in catalytic activity when compared with wild-type (WT) enzyme (～ 7.4-fold decrease in k(cat) for the C13D and ～ 3.3-fold for the C13E mutants). Analytical gel filtration demonstrates that the C13D mutant dissociates at concentrations <1.25 μM, whereas the WT and the C13E enzymes retain the dimeric structure. The order of stability of the mutants in the presence of chemical denaturants, like urea and guanidium chloride, is WT > Cys13Glu > Cys13Asp. Irreversible thermal precipitation temperatures follow the same order as well. Modeling studies establish that the Cys13Asp mutation is likely to cause a significantly greater structural perturbation than Cys13Glu. Analysis of sequence and structural data for TIMs from diverse sources suggests that residues 13 and 82 form a pair of proximal sites, in which a limited number of residue pairs may be accommodated.     

Roles of catalytic residues in {alpha}-amylases as evidenced by the structures of the product-complexed mutants of a maltotetraose-forming amylase

The crystal structures of the four product-complexed singlemutants of the catalytic residues of Pseudomonas stutzeri maltotetraose-forming-amylase, E219G, D193N, D193G and D294N, have been determined.Possible roles of the catalytic residues Glu219, Asp193 andAsp294 have been discussed by comparing the structures amongthe previously determined complexed mutant E219Q and the presentmutant enzymes. The results suggested that Asp193 predominantlyworks as the base catalyst (nucleophile), whose side chain atomlies in close proximity to the C1-atom of Glc4, being involvedin the intermediate formation in the hydrolysis reaction. WhileAsp294 works for tightly binding the substrate to give a twistedand a deformed conformation of the glucose ring at position–1 (Glc4). The hydrogen bond between the side chain atomof Glu219 and the O1-atom of Glc4, that implies the possibilityof interaction via hydrogen, consistently present throughoutthese analyses, supports the generally accepted role of thisresidue as the acid catalyst (proton donor).     

Physicochemical Methods for Prediction of Functional Information for Proteins

Structural genomics initiatives are determining thousands of new protein structures. Many of these structures are of unknown function, and computational methods for the rapid determination of functional information from protein structure are needed. We present details of how functional information is obtained from the structure using THEMATICS (Theoretical Microscopic Titration Curves). THEMATICS is a computational procedure that gives information about chemical reactivity, based on solution of the Poisson-Boltzmann equations for the electrical potential function. We show how anomalies in predicted titration curves are established. We show further that when residues with anomalous predicted titration curves form a cluster in physical space, these residues tend to be very highly conserved across species and such clusters are reliable predictors of the active site. Results are given for ten enzymes; detailed results are shown for the enzymes triosephosphate isomerase (from chicken), 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (from E. coli), and papain (from papaya).