Proton magnetic resonance studies of ribonuclease T1. Assignment of histidine-40 peak and analysis of the active site.

Nuclear magnetic resonances of the C-2 protons of the three histidine residues in ribonuclease T₁ have been studied at 360 MHz as a function of pH to discuss the structure of the active site. Comparison of the order of deuterium exchange of the histidine peaks with tritium incorporation rates into individual histidines of the enzyme leads to the unambigous assignment of one of the C-2 proton peaks to histidne-40. It has been concluded that histidine-40 is in the active site, interacting with a charged group of pK 4.1, which is replaced by the phosphate group of guanosine-3′-monophosphate in the enzyme-inhibitor complex. Histidine-92 is most likely a binding site for the complex, where the existence of a hydrogen bond between N-7 of the inhibitor and the ring NH proton of the histidine is suggested on the basis of NMR data.     

Correlation of exchangeable (NH) and nonexchangeable (C2H) histidine resonances in the proton NMR spectrum of ribonuclease A in aqueous solution.

The ionization characteristics of the hydrogen-bonded His 12 N₁ proton observed to titrate between 11 to 13 ppm in the nmr spectrum of ribonuclease A in H₂O solution are compared with the ionization characteristics of the four histidine C₂ protons in the enzyme. Comparison of the pK_a's of the enzyme in H₂O and D₂O in the absence and presence of cytidine monophosphate (?5′, ?3′, and ?2′) inhibitors, line widths in the presence of Cu II at pH 3.6 and 5.6, and chemical shifts in the presence of AgNO₃ permit a correlation of the exchangeable His 12 N₁ proton with the active site histidine C₂ proton exhibiting the lower ionization pK_a. The histidines with pK_a of 5.1 and 5.6 in ribonuclease A in the absence of salt are assigned in this study to His 12 and His 119, respectively.     

A study of the histidine residues of human carbonic anhydrase B using 270 MHz proton magnetic resonance

Nine resonances in the 270 MHz proton magnetic resonance spectrum of human carbonic anhydrase B have been identified with imidazole C₍₂₎ protons of histidine residues, six of which are observed to titrate with pK_a values in the range 4.7 to 7.4. The behaviour of the nine resonances has been studied in the presence of the inhibitors, iodide, cyanide, acetate, hexacyanochromate, and imidazole. Measurements have also been made of the enzyme in its apo, cobalt, and mono-alkylated forms. Used in conjunction with the crystal structure, these results have enabled the tentative assignment of all nine resonances to particular histidine residues in the amino-acid sequence. Three of the active-site histidines at positions 64, 67, and 200 have low pK_a values and cannot be directly linked to the activity of the enzyme. However, the resonances assigned to the three metal-liganding histidines do exhibit changes on anion binding and with pH, which parallel changes in the esterase activity. These results are consistent with the model of an ionizable water molecule bound to the zinc ion.Linewidth measurements of the resonances of the histidine residues on the enzyme surface are used to estimate pseudo-first-order rate constants of the order of 4 × 10³ s^?1 for D⁺ exchange between imidazole N and solvent in the absence of buffer. These rates are observed to increase in the presence of small amounts of the buffers Tris and imidazole.     

Evidence for two variants of poly(adenosine diphosphate ribose) glycohydrolase in rat testis.

The specific activity of rat poly(adenosine diphosphate ribose) glycohydrolase was higher in the testis than in the liver, brain, spleen or kidney. The enzyme was found primarily in the soluble fraction of the testis. When the soluble enzyme was chromatographed on phosphocellulose, the activity eluted in two peaks, at 0.22 and 0.34 m KCl, respectively, referred to in the present study as enzyme A and B. Enzyme A has an optimal pH of 7.25 and was stimulated by 150 mm KCl. The optimal pH of enyzme B was 6.5, but it was not stimulated by KCl. For maximal activity both enzymes required 10 mm 2-mercaptoethanol, and they were strongly inhibited by 100 μmp-chloromercuribenzoate. The K_m values of enzyme A and B for poly(adenosine diphosphate ribose) were 1.52 and 0.70 μm, respectively. Ribose 5′-phosphate, guanosine 3′,5′-monophosphate, adenosine 3′,5′-monophosphate and adenosine diphosphate ribose inhibited both enzymes. The two latter nucleotides behave as noncompetitive inhibitors. Denatured DNA and the homopolypurines poly(G), poly(I) and poly(A) were very potent inhibitors of both glycohydrolases. The mode of hydrolysis of poly(adenosine diphosphate ribose) by glycohydrolases A and B was exoglycosidic, yielding adenosine diphosphate ribose as the final product.     

Correlation proton magnetic resonance studies at 250 MHz of bovine pancreatic ribonuclease. I. Reinvestigation of the histidine peak assignments.

The deuterium exchange kinetics of the C(2) protons of the four histidine residues of native bovine pancreatic ribonuclease A have been followed at pH 6.5 and 8.0 by proton magnetic resonance spectroscopy (1H NMR). Comparison of the order of exchange of the histidine peaks with tritium exchange rates into individual histidine residues [Ohe, M., Matsuo, H., Sakiyama, F., and Narita, K. (1974), J. Biochem. (Tokyo) 75, 1197] supports the previous assignment of histidine NMR peaks H(1) and H(4) to histidine-105 and histidine-48 but requires reassignment of peaks H(2) and H(3) to histidine-119 and histidine-12, respectively. Ribonuclease A samples having differentially deuterated histidines have been used to verify the existence of crossover points in the histidine proton magnetic resonance titration curves and to observe the discontinuous titration curve of histidine-48. Proton magnetic resonance peaks have been assigned to the C(4) protons of the four histidine residues of ribonuclease A on the basis of their unit proton areas and by matching their titration shifts with the more readily visible C(2)-H peaks of the histidines. The pK' values derived from the C(4)-H data agree, within experimental limits, with those derived from C(2)-H data. The C(4)-H peaks were assigned to histidine-12, -48, -105, and -119 of ribonuclease A on the basis of their pH dependence, pK' values, shifts of their pK' values in the presence of inhibitor cytidine 3'-phosphate, and by comparison with the assignments of the histidine C(2)-H peaks above.     

The synthesis of choline and ethanolamine phosphoglycerides in neuronal and glial cells of rabbit in vitro

Abstract— The de novo synthesis of phosphatidylcholine and phosphatidylethanolamine in isolated neuronal and glial cells from adult rabbit brain cortex was investigated in vitro, using labelled phosphorylcholine (phosphorylethanolamine) or cytidine-5′-phosphate choline (cytidine-5′-phosphate ethanolamine), as lipid precursors. Synthesis of phospholipid from phosphorylcholine and phosphorylethanolamine in both fractions was extremely low when compared to that derived from the corresponding cytidine nucleotides. The neuronal cell-enriched fraction was found to possess a much higher rate of synthesis of both lipids from all precursors. Neuronal/glial ratios of about 5–9 were found for the synthesis of phosphatidylcholine and phosphatidylethanolamine from cytidine-5′-phosphate choline and cytidine-5′-phosphate ethanolamine, respectively. Several kinetic properties of the choline-phosphotransferase (EC 2.7.8.2) and ethanolaminephosphotransferase (EC 2.7.8.1) were found to be similar both in neurons and in glia (e.g. K_m of cytidine-5′-phosphate ethanolamine, K_m of diacyl glycerol, pH optimum, need for divalent cations), but the K_m value for cytidine-5′-phosphate choline in glial cells was much lower (2.3 × 10^?4m ) than in neurons (1 × 10^?3m ). The K_mfor cytidine-5′-phosphate ethanolamine in both cells was much lower than in whole brain microsomes. It is concluded that the cytidine-dependent enzymic system for phosphatidylcholine and phosphatidylethanolamine synthesis is concentrated mostly in the neuronal cells, as compared to glia.     

Reassignment of the active site histidines in ribonuclease A by selective deuteration studies.

The C₂H resonance of the active site histidine residue designated AS-2, which has the lower pK_a of the two active site histidines, has been correlated in both RNase A and RNase S by comparing the pH 3 to 5.5 regions of the chemical shift titration curves, the effect of the inhibitor CMP-3′ on the chemical shifts at pH 4.0, and the effect of Cu II on the line widths at pH 3.6. It has been demonstrated that resonance AS-2 is absent in the spectrum of RNase S′ reconstituted using S-peptide deuterated at the C₂ of His 12, and in that of the RNase S′-CMP-3′ complex. We thus demonstrate that histidine AS-2 is in fact His 12 in both enzymes. This finding is in agreement with out previous assignment of the exchangeable NH proton in RNase A to His 12, but reverses the assignments of the active site histidine C₂H resonances made earlier by other authors.     

Nuclear magnetic resonance titration curves of histidine ring protons. A direct assignment of the resonances of the active site histidine residues of ribonuclease.

One of the four titrating histidine ring C-2 proton resonances of bovine pancreatic ribonuclease has been assigned to histidine residue 12. This was accomplished by a direct comparison of the rate of tritium incorporation into position C-2 of histidine 12 of S-peptide (residues 1 to 20) derived from ribonuclease S, with the rates of deuterium exchange of the four histidine C-2 proton resonances of ribonuclease S under the same experimental conditions. The same assignment was obtained by a comparison of the NMR titration curves of ribonuclease S, the noncovalent complex of S-peptide and S-protein (residues 21 to 124) with the results for the recombined complex in which position C-2 of histidine 12 was fully deuterated. The second active site histidine resonance was assigned to histidine residue 119 by consideration of the NMR titration results fro carboxymethylated histidines and 1-carboxymethylhistidine 119 ribonuclease. This assignment is a reversal of that originally reported, and has important implications for the interpretation of NMR titration data of ribonuclease.     

Comparative studies of the interaction of human and bovine platelet factor 4 with heparin using histidine NMR resonances as spectroscopic probes

ThepK _a values of His-38 and His-50 of the heparin-binding protein, bovine platelet factor 4, are 5.6 and 6.5, respectively, as determined by¹H NMR spectroscopy. The¹H NMR resonance of His-38 of bovine platelet factor 4 which exhibits the lowerpK _a value is perturbed upon heparin binding to a greater degree than the resonance of His-50. Human platelet factor 4 contains the homologous residues His-23 and His-35. ThepK _a values of the two histidine residues of human platelet factor 4 are 5.3 and 6.4. The¹H NMR resonance of the histidine of human platelet factor 4 exhibiting the lowerpK _a value also is perturbed upon heparin binding to a greater degree than the histidine resonance exhibiting the higherpK _a , thereby suggesting comparable heparin-protein interactions in bovine and human platelet factor 4.     

Analysis of essential histidine residues of maize branching enzymes by chemical modification and site-directed mutagenesis

Incubation of maize branching enzyme, mBEI and mBEII, with 100 μM diethylpyrocarbonate (DEPC) rapidly inactivated the enzymes. Treatment of the DEPC-inactivated enzymes with 100–500 mM hydroxylamine restored the enzyme activities. Spectroscopic data indicated that the inactivation of BE with DEPC was the result of histidine modification. The addition of the substrate amylose or amylopectin retarded the enzyme inactivation by DEPC, suggesting that the histidine residues are important for substrate binding. In maize BEII, conserved histidine residues are in catalytic regions 1 (His320) and 4 (His508). His320 and His508 were individually replaced by Ala via site-directed mutagenesis to probe their role in catalysis. Expression of these mutants inE. coli showed a significant decrease of the activity and the mutant enzymes hadK _m values 10 times higher than the wild type. Therefore, residues His320 and His508 do play an important role in substrate binding.     

Proton nuclear magnetic resonance studies on bovine lutropin, its subunits, and on the alpha subunit of pregnant mare serum gonadotropin. Assignment of histidine resonances in the alpha subunit.

The pK values of the 3 histidine residues in the common alpha subunits of bovine and equine glycoprotein hormones have been determined from titration curves generated from their C-2 proton nuclear magnetic resonances at different pH values. Assignment of resonances to specific histidines is based on a comparison between the two species, which have 1 histidine residue in different positions in their sequences, and of the bovine alpha subunit after removal of its histidine 94 by treatment with carboxypeptidases. In both species, those histidines closest to the COOH terminus titrate with near normal pK values of 6.2. The histidine residue found in the bovine subunit at position 87 titrates with an approximate pK value of 5.4. Histidine 83, adjacent to an oligosaccharide moiety in both species, does not titrate over a pH range of 4.0 to 8.0 and thus appears inaccessible to solvent. Similarly, in bovine lutropin-beta, 1 of 3 histidine residues does not titrate between pH 5.0 and 7.0. In the intact hormone, 2 "nontitratable" histidine residues are found. Changes in the characteristics of the signals, however, preclude unambiguous assignment of these two resonances to the nontitrating histidines in the isolated subunits. It appears that changes in the environment of at least some histidines occur when the subunits combine to yield intact hormone.     

Effect of Diethylpyrocarbonate on the Allosteric Properties of Phosphoenolpyruvate Carboxylase from Crassula argentea

Phosphoenolpyruvate carboxylase from the Crassulacean acid metabolism plant Crassula argentea was substantially desensitized to the effects of regulatory ligands by treatment with diethylpyrocarbonate, a reagent which selectively modifies histidyl residues. Desensitization of the enzyme to the inhibitor malate and the activator glucose 6-phosphate was accompanied by the appearance of a peak in the ultraviolet difference spectrum at 240 nanometers, indicating the formation of ethoxyformylhistidyl derivatives. Hydroxylamine reversed part of the spectral change under native conditions, and almost all of the change under denaturing conditions, but failed to restore sensitivity to effectors. The pH profiles of desensitization to malate and glucose 6-phosphate indicated the involvement of groups on the enzyme with pK, values of 6.8 and 6.4, respectively. Under denaturing conditions, a total of 15 histidine residues per subunit were modified by diethylpyrocarbonate, whereas for the native enzyme nine histidines were modified per subunit. Effector desensitization occurs after the modification of two to three histidyl residues per subunit. The presence of malate reduced the apparent rate constant for desensitization by 60%, suggesting that the modification occurred at the malate binding site. Diethylpyrocarbonate treatment also eliminated the kinetic lag caused by malate. Glucose 6-phosphate did not protect the enzyme against diethylpyrocarbonate-induced desensitization.     

A physical difference between the fast- and slow-refolding forms of nitrotyrosyl ribonuclease A: the pK values of the nitrotyrosyl groups.

In the preceding paper we present kinetic evidence for a slow equilibrium between two conformational forms of heat-unfolded ribonuclease A whose rates of refolding differ 100-fold. In a search for physical differences between these two forms, we undertook a study of the pK changes during refolding of a specific set of freely ionizing surface groups. By use of a standard procedure the three freely ionizing tyrosine groups (pK ∼- 10) have been nitrated by tetranitromethane, yielding three nitrotyrosine groups (pK ∼- 6.8). Nitrotyrosyl ribonuclease A closely resembles the unmodified enzyme as regards: (1) enzymatic activity; (2) thermal unfolding transition at neutral pH; and (3) kinetics of refolding. In particular, stopped-flow measurements of 2′CMP binding during refolding show that the fast-refolding reaction is unchanged by nitration and yields fully folded enzyme able to bind 2′CMP.The pK change of the nitrotyrosyl groups upon refolding is quite different in the fast- and slow-refolding reactions. In the slow reaction it is small (− 0.046 ± 0.006 pH unit) but easily measureable, whereas in the fast-reaction it is too small to be detected (− ΔpK less than 0.02 pH unit). This difference in pK change upon refolding can be attributed to different pK values of the nitrotyrosyl groups in the slow-refolding and fast-refolding forms of the heat-unfolded protein. Presumably the same structural differences between these two heat-unfolded forms are responsible both for the pK difference and for the 100-fold difference in rates of refolding.These results support the simple three-species mechanism for refolding discussed in the preceding paper. (a) They demonstrate a physical difference between the fast- and slow-refolding species. (b) They do not show any additional kinetic complexity when refolding is measured by a property that distinguishes between the fast- and slow-refolding species.     

Histidine hydrogen-deuterium exchange mass spectrometry for probing the microenvironment of histidine residues in dihydrofolate reductase

Background

Histidine Hydrogen-Deuterium Exchange Mass Spectrometry (His-HDX-MS) determines the HDX rates at the imidazole C₂-hydrogen of histidine residues. This method provides not only the HDX rates but also the pK _a values of histidine imidazole rings. His-HDX-MS was used to probe the microenvironment of histidine residues of E. coli dihydrofolate reductase (DHFR), an enzyme proposed to undergo multiple conformational changes during catalysis.

Methodology/Principal Findings

Using His-HDX-MS, the pK _a values and the half-lives (t _1/2) of HDX reactions of five histidine residues of apo-DHFR, DHFR in complex with methotrexate (DHFR-MTX), DHFR in complex with MTX and NADPH (DHFR-MTX-NADPH), and DHFR in complex with folate and NADP⁺ (DHFR-folate-NADP⁺) were determined. The results showed that the two parameters (pK _a and t _1/2) are sensitive to the changes of the microenvironment around the histidine residues. Although four of the five histidine residues are located far from the active site, ligand binding affected their pK _a, t _1/2 or both. This is consistent with previous observations of ligand binding-induced distal conformational changes on DHFR. Most of the observed pK _a and t _1/2 changes could be rationalized using the X-ray structures of apo-DHFR, DHFR-MTX-NADPH, and DHFR-folate-NADP⁺. The availability of the neutron diffraction structure of DHFR-MTX enabled us to compare the protonation states of histidine imidazole rings.

Conclusions/Significance

Our results demonstrate the usefulness of His-HDX-MS in probing the microenvironments of histidine residues within proteins.     

The pKa values of two histidine residues in human haemoglobin, the Bohr effect, and the dipole moments of alpha-helices

Studies of abnormal and chemically modified haemoglobins indicate that in 0.1 m-NaCl about 40% of the alkaline Bohr effect of human haemoglobin is contributed by the C-terminal histidine HC3(146)β. In deoxyhaemoglobin, the imidazole of this histidine forms a salt bridge with aspartate FG1(94)β, in oxyhaemoglobin or carbonmonoxyhaemoglobin it accepts a hydrogen bond from its own NH group instead. Kilmartin et al. (1973) showed that in 0.2 m-NaCl + 0.2 m-phosphate this change of ligation lowered the pK_a of the histidine from 8.0 in Hb³ to 7.1 in HbCO, but Russu et al. (1980) claimed that in bis-Tris buffer without added NaCl its pK_a in HbCO dropped no lower than 7.85, and that in this medium the C-terminal histidine made only a negligible contribution to the alkaline Bohr effect.We have compared the histidine resonances of HbCO A with those of three abnormal haemoglobins: HbCO Cowtown (His HC3(146)β → Leu), HbCO Wood (His FG4(97)β → Leu) and HbCO Malmø (His FG4(97)β → Gln). Our results show that the resonance assigned by Russu et al. to His HC3(146)β in fact belongs to His FG4(97)β. Although in Hb the pK_a of His HC3(146)β is 8.05 ± 0.05 independent of ionic strength, in HbCO its pK_a drops sharply with diminishing ionic strength, so that in the buffer employed by Russu et al. it has a pK_a of 6.2 and makes a contribution to the alkaline Bohr effect that is 57% larger than in the phosphate buffer employed by Kilmartin et al. (1973).In HbCO A, His FG4(97)β does not contribute to the Bohr effect, but in HbCO from which His HC3(146)β has been cleaved (HbCO des-His), His FG4(97)β is in equilibrium between two conformations with different pK_a values. This equilibrium varies with ionic strength and pH, and presumably also with degree of ligation of the haem moiety.In HbCO A, His FG4(97)β has a pK_a of 7.8 compared to the pK_a value of about 6.6 characteristic of free histidines at the surface of proteins. This high pK_a is accounted for by its interaction with the negative pole at the C terminus of helices F and FG. It corresponds to a free energy change of the same order as that observed in the interaction of histidines with carboxylate ions and confirms the strongly dipolar character of α-helices, which manifests itself even when they lie on the surface of the protein.     

Proton magnetic resonance study of 8-(6-aminohexyl)-aminoadenosine 5′-monophosphate

The nucleotide 8-(6-aminohexyl)-amino adenosine 5′-monophosphate (8-AHA-AMP) has been investigated by 220-MHz proton magnetic resonance spectroscopy. The conformation and ionization state of the nucleotide have been determined. The anti-conformation about the glycosyl bond is the preferred form. The interaction between the hexyldiamino chain and the ribose moiety in this conformation gives rise to unusual ribosyl conformation results. The distribution of conformations about the glycosyl bond has little influence on the effectiveness of this nucleotide analog in the purification of dehydrogenases by affinity chromatography. The chemical shift dependence on pH has been carried out on 8-methylaminoadenosine 5′-monophosphate. The 8-aminoadenine ring is protonated at N1 (pK_α 5.0) and at N7 (pK_α 1.5) in acidic solutions. The protonation at N7 is apparently stabilized by a delocalization of charge onto the 8-amino group. The neutrality of the 8-aminoadenine ring at physiological pH is consistent with the effcient binding of the nucleotide by dehydrogenases. An improved method for the preparation of the 8-AHA-AMP is described.     

The charge-relay system of serine proteinases: proton magnetic resonance titration studies of the four histidines of porcine trypsin.

Peaks corresponding to the C₍₂₎-protons of all four histidine residues of porcine β-trypsin were resolved in 250 MHz nuclear magnetic resonance spectra after deuteration of the slowly exchangeable N-H groups (whose resonances obscure the histidine peaks) by reversible unfolding of the protein in D₂O. One of the four peaks was assigned to the charge-relay histidine in the active site of trypsin (His(57) in the bovine chymotrypsinogen numbering system). Whereas the three other histidine C₍₂₎-peaks exhibited normal titration curves with single pK′ values of 7.20, 6.71 and 6.67, the peak assigned to His(57) had an abnormal titration curve showing two protonation steps in the pH range from 1 to 9. The first protonation with a pH′_mid of 5.0 is rapid on the nuclear magnetic resonance time-scale; the second with a pH′_mid of 4.5 is slow and apparently involves conformational transitions between two states having lifetimes of approximately 18 ms.In the complex between porcine β-trypsin and bovine pancreatic trypsin inhibitor (Kunitz) His(57) was found to be insensitive to pH over the range from 4 to 9 and its chemical shift resembles that of His(57) in the singly protonated charge relay of free trypsin. This result provides direct evidence that the trypsin charge relay acts as a proton acceptor in the initial catalytic step which leads to the formation of a tetrahedral complex. In the presence of equimolar bovine pancreatic trypsin inhibitor (Kunitz) the pH'_mid of the conformational transition that affects the charge-relay histidine is lowered from 4.5 to approximately 3.5.     

Evidence linking the Pseudomonas oleovorans alkane omega-hydroxylase,an integral membrane diiron enzyme,and the fatty acid desaturase family

Pseudomonas oleovorans alkane omega-hydroxylase (AlkB) is an integral membrane diiron enzyme that shares a requirement for iron and oxygen for activity in a manner similar to that of the non-heme integral membrane desaturases, epoxidases, acetylenases, conjugases, ketolases, decarbonylase and methyl oxidases. No overall sequence similarity is detected between AlkB and these desaturase-like enzymes by computer algorithms; however, they do contain a series of histidine residues in a similar relative positioning with respect to hydrophobic regions thought to be transmembrane domains. To test whether these conserved histidine residues are functionally equivalent to those of the desaturase-like enzymes we used scanning alanine mutagenesis to test if they are essential for activity of AlkB. These experiments show that alanine substitution of any of the eight conserved histidines results in complete inactivation, whereas replacement of three non-conserved histidines in close proximity to the conserved residues, results in only partial inactivation. These data provide the first experimental support for the hypotheses: (i) that the histidine motif in AlkB is equivalent to that in the desaturase-like enzymes and (ii) that the conserved histidine residues play a vital role such as coordinating the Fe ions comprising the diiron active site.     

The role of histidine residues of formate dehydrogenase from Bacterium sp. 1]

The reaction between formate dehydrogenase from Bacterium sp. 1 and diethylpyrocarbonate results in the enzyme inactivation. 4 histidine residues can be blocked per subunit by this reagent. The enzyme activity correlates with the disappearance of free histidines. The process of enzyme inactivation is biphasic and obeys pseudo-first-order kinetics. NAD and NADH slow down the rate of inactivation, but do not protect histidine residues against modification. Formate does not protect the enzyme. The modification of 80% of histidines increases the Km value for both substrates 3-fold. The general conformation of enzyme in the course of modification is preserved. The modification of histidines markedly decreases the reactivity of an essential SH-group of formate dehydrogenase against the Ellman reagent.     

Mutation of non-conserved amino acids surrounding catalytic site to shift pH optimum of Bacillus circulans xylanase

Improvement of enzyme function by engineering pH dependence of enzymatic activity is of importance for industrial application of Bacillus circulans xylanases. Target mutation sites were selected by structural alignment between B. circulans xylanase and other xylanases having different pH optima. We selected non-conserved mutant sites within 8 Å from the catalytic residues, to see whether these residues have some role in modulating pK_as of the catalytic residues. We hypothesized that the non-conserved residues which may not have any role in enzyme catalysis might perturb pK_as of the catalytic residues. Change in pK_a of a titratable group due to change in electrostatic potential of a mutation was calculated and the change in pH optimum was predicted from the change in pK_a of the catalytic residues. Our strategy is proved to be useful in selection of promising mutants to shift the pH optimum of the xylanases towards desired side.