Analysis of human ITPase nucleobase specificity by site-directed mutagenesis

Inosine triphosphate (ITP) pyrophosphohydrolase, or ITPase, is an intracellular enzyme that is responsible for the hydrolysis of the acidic anhydride bond between the alpha and beta phosphates in ITP, and other noncanonical nucleoside triphosphates, producing the corresponding nucleoside monophosphate and pyrophosphate. This activity protects the cell by preventing noncanonical nucleoside triphosphates from accumulating in (deoxy) nucleoside triphosphate ((d)NTP) pools and/or being integrated into nucleic acids. This enzyme is encoded by the ITPA gene in mammals. It has been reported that Itpa homozygous-null knock-out mice die before weaning and have gross cardiac abnormalities. Additionally, certain variations in the human ITPA gene have been linked to adverse reactions to the immunosuppressive prodrugs azathioprine and 6-mercaptopurine and protection against ribavirin-induced hemolytic anemia. These drugs are bioactivated to form noncanonical nucleoside triphosphates. Human ITPase enzymes engineered to modulate nucleobase specificity may be valuable tools for studying the role of ITPase in heart development and drug metabolism or developing gain-of-function mutants or inhibitory molecules. Based on x-ray crystallography and amino acid sequence data, a panel of putative human ITPase nucleobase specificity mutants has been generated. We targeted eight highly conserved amino acid positions within the ITPase sequence that correspond to amino acids predicted to directly interact with the nucleobase or help organize the nucleobase binding pocket. The ability of the mutants to protect against exogenous and endogenous noncanonical purines was tested with two Escherichia coli complementation assays. Nucleobase specificity of the mutants was investigated with an in vitro biochemical assay using ITP, GTP and ATP as substrates. This methodology allowed us to identify gain-of-function mutants and categorize the eight amino acid positions according to their ability to protect against noncanonical purines as follows: Glu-22, Trp-151 and Arg-178, essential for protection; Phe-149, Asp-152, Lys-172 and Ser-176, intermediate protection; His-177, dispensable for protection against noncanonical purines.     

Yeast methionine aminopeptidase I. Alteration of substrate specificity by site-directed mutagenesis

In eukaryotes, two isozymes (I and II) of methionine aminopeptidase (MetAP) catalyze the removal of the initiator methionine if the penultimate residue has a small radius of gyration (glycine, alanine, serine, threonine, proline, valine, and cysteine). Using site-directed mutagenesis, recombinant yeast MetAP I derivatives that are able to cleave N-terminal methionine from substrates that have larger penultimate residues have been expressed. A Met to Ala change at 329 (Met206 in Escherichia coli enzyme) produces an average catalytic efficiency 1.5-fold higher than the native enzyme on normal substrates and cleaves substrates containing penultimate asparagine, glutamine, isoleucine, leucine, methionine, and phenylalanine. Interestingly, the native enzyme also has significant activity with the asparagine peptide not previously identified as a substrate. Mutation of Gln356 (Gln233 in E. coli MetAP) to alanine results in a catalytic efficiency about one-third that of native with normal substrates but which can cleave methionine from substrates with penultimate histidine, asparagine, glutamine, leucine, methionine, phenylalanine, and tryptophan. Mutation of Ser195 to alanine had no effect on substrate specificity. None of the altered enzymes produced cleaved substrates with a fully charged residue (lysine, arginine, aspartic acid, or glutamic acid) or tyrosine in the penultimate position.     

In vivo analysis of the Saccharomyces cerevisiae HO nuclease recognition site by site-directed mutagenesis.

HO nuclease introduces a specific double-strand break in the mating-type locus (MAT) of Saccharomyces cerevisiae, initiating mating-type interconversion. To define the sequence recognized by HO nuclease, random mutations were produced in a 30-base-pair region homologous to either MAT alpha or MATa by a chemical synthesis procedure. The mutant sites were introduced into S. cerevisiae on a shuttle vector and tested for the ability to stimulate recombination in an assay that mimics mating-type interconversion. The results suggest that a core of 8 noncontiguous bases near the Y-Z junction of MAT is essential for HO nuclease to bind and cleave its recognition site. Other contacts must be required because substrates that contain several mutations outside an intact core reduce or eliminate cleavage in vivo. The results show that HO site recognition is a complex phenomenon, similar to promoter-polymerase interactions.     

Engineering of papain: selective alteration of substrate specificity by site-directed mutagenesis

The S2 subsite specificity of the plant protease papain has been altered to resemble that of mammalian cathepsin B by site-directed mutagenesis. On the basis of amino acid sequence alignments for papain and cathepsin B, a double mutant (Val133Ala/Ser205Glu) was produced where Val133 and Ser205 are replaced by Ala and Glu, respectively, as well as a triple mutant (Val133Ala/Val157Gly/Ser205Glu), where Val157 is also replaced by Gly. Three synthetic substrates were used for the kinetic characterization of the mutants, as well as wild-type papain and cathepsin B: CBZ-Phe-Arg-MCA, CBZ-Arg-Arg-MCA, and CBZ-Cit-Arg-MCA. The ratio of kcat/KM obtained by using CBZ-Phe-Arg-MCA as substrate over that obtained with CBZ-Arg-Arg-MCA is 8.0 for the Val133Ala/Ser205Glu variant, while the equivalent values for wild-type papain and cathepsin B are 904 and 3.6, respectively. This change in specificity has been achieved by replacing only two amino acids out of a total of 212 in papain and with little loss in overall enzyme activity. However, further replacement of Val157 by Gly as in Val133Ala/Val157Gly/Ser205Glu causes an important decrease in activity, although the enzyme still displays a cathepsin B like substrate specificity. In addition, the pH dependence of activity for the Val133Ala/Ser205Glu variant compares well with that of cathepsin B. In particular, the activity toward CBZ-Arg-Arg-MCA is modulated by a group with a pKa of 5.51, a behavior that is also encountered in the case of cathepsin B but is absent with papain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alteration of substrate specificity of leucine dehydrogenase by site-directed mutagenesis

The residues L40, A113, V291, and V294, in leucine dehydrogenase (LeuDH), predicted to be involved in recognition of the substrate side chain, have been mutated on the basis of the molecular modeling to mimic the substrate specificities of phenylalanine (PheDH), glutamate (GluDH), and lysine dehydrogenases (LysDH). The A113G and A113G/V291L mutants, imitating the PheDH active site, displayed activities toward -phenylalanine and phenylpyruvate with 1.6 and 7.8% of k_cat values of the wild-type enzyme for the preferred substrates, -leucine and its keto-analog, respectively. Indeed, the residue A113, corresponding to G114 in PheDH, affects the volume of the side-chain binding pocket and has a critical role in discrimination of the bulkiness of the side chain. Another two sets of mutants, substituting L40 and V294 of LeuDH with the corresponding residues predicted in GluDH and LysDH, were also constructed and characterized. Emergence of GluDH and LysDH activities in L40K/V294S and L40D/V294S mutants, respectively, indicates that the two corresponding residues in the active site of amino acid dehydrogenases are important for discrimination of the hydrophobicity/polarity of the aliphatic substrate side chain. All these results demonstrate that the substrate specificities of the amino acid dehydrogenases can be altered by protein engineering. The engineered dehydrogenases are expected to be used for production and detection of natural and non-natural amino acids.     

Identification of residues involved in v-Src substrate recognition by site-directed mutagenesis.

To study the role of the catalytic domain in v-Src substrate specificity, we engineered three site-directed mutants (Leu-472 to Tyr or Trp and Thr-429 to Met). The mutant forms of Src were expressed in Sf9 cells and purified. We analyzed the substrate specificities of wild-type v-Src and the mutants using two series of peptides that varied at residues C-terminal to tyrosine. The peptides contained either the YMTM motif found in insulin receptor substrate-1 (IRS-1) or the YGEF motif identified from peptide library experiments to be the optimal sequence for Src. Mutations at positions Leu-472 or Thr-429 caused changes in substrate specificity at positions P+1 and P+3 (i.e. one or three residues C-terminal to tyrosine). This was particularly evident in the case of the L-472W mutant, which had pronounced alterations in its preferences at the P+1 position. The results suggest that residue Leu-472 plays a role in P+1 substrate recognition by Src. We discuss the results in the light of recent work on the roles of the SH2, SH3 and catalytic domains of Src in substrate specificity.     

Alteration of substrate specificity of cholesterol oxidase from Streptomyces sp. by site-directed mutagenesis

Despite the structural similarities between cholesterol oxidase from Streptomyces and that from Brevibacterium, both enzymes exhibit different characteristics, such as catalytic activity, optimum pH and temperature. In attempts to define the molecular basis of differences in catalytic activity or stability, substitutions at six amino acid residues were introduced into cholesterol oxidase using site-directed mutagenesis of its gene. The amino acid substitutions chosen were based on structural comparisons of cholesterol oxidases from Streptomyces and BREVIBACTERIUM: Seven mutant enzymes were constructed with the following amino acid substitutions: L117P, L119A, L119F, V145Q, Q286R, P357N and S379T. All the mutant enzymes exhibited activity with the exception of that with the L117P mutation. The resulting V145Q mutant enzyme has low activities for all substrates examined and the S379T mutant enzyme showed markedly altered substrate specificity compared with the wild-type enzyme. To evaluate the role of V145 and S379 residues in the reaction, mutants with two additional substitutions in V145 and four in S379 were constructed. The mutant enzymes created by the replacement of V145 by Asp and Glu had much lower catalytic efficiency for cholesterol and pregnenolone as substrates than the wild-type enzyme. From previous studies and this study, the V145 residue seems to be important for the stability and substrate binding of the cholesterol oxidase. In contrast, the catalytic efficiencies (k(cat)/K(m)) of the S379T mutant enzyme for cholesterol and pregnenolone were 1.8- and 6.0-fold higher, respectively, than those of the wild-type enzyme. The enhanced catalytic efficiency of the S379T mutant enzyme for pregnenolone was due to a slightly high k(cat) value and a low K(m) value. These findings will provide several ideas for the design of more powerful enzymes that can be applied to clinical determination of serum cholesterol levels and as sterol probes.     

Alteration of the binding specificity of cellular retinol-binding protein II by site-directed mutagenesis.

Rat cellular retinol-binding protein II (CRBP II) is an abundant 134-residue intestinal protein that binds all-trans-retinol and all-trans-retinal. It belongs to a family of homologous, 15-kDa cytoplasmic proteins that bind hydrophobic ligands in a noncovalent fashion. These binding proteins include a number of proteins that bind long chain fatty acids. X-ray analyses of the structure of two family members, rat intestinal fatty acid-binding protein and bovine myelin P2 protein, indicate that they have a high degree of conformational similarity and that the carboxylate group of their bound fatty acid interacts with a delta-guanidium group of at least 1 of 2 "buried" arginine residues. These 2 Arg residues are conserved in other family members that bind long chain fatty acids and in cellular retinoic acid-binding protein, but are replaced by Gln109 and Gln129 in CRBP II. We have genetically engineered two amino acid substitutions in CRBP II: 1) Gln109 to Arg and 2) Gln129 to Arg. The purified Escherichia coli-derived CRBP II mutant proteins were analyzed by fluorescence and nuclear magnetic resonance spectroscopy. Both mutants exhibit markedly decreased binding of all-trans-retinol and all-trans-retinaldehyde, but no increased binding of all-trans-retinoic acid. Arg substitution for Gln109 but not for Gln129 produces a dramatic increase in palmitate binding activity. Analysis of the endogenous fatty acids associated with the purified E. coli-derived proteins revealed that E. coli-derived intestinal fatty acid binding protein and the Arg109 CRBP II mutant are complexed with endogenous fatty acids in a qualitatively and quantitatively similar manner. These results provide evidence that this internal Arg may play an important role in the binding of long chain fatty acids by members of this protein family.     

Functional analysis of ISC1 by site-directed mutagenesis

We previously reported that the yeast Saccharomyces cerevisiae ISC1 gene (Yer019w), which has homology to the bacterial sphingomyelinase gene, encodes inositol phosphosphingolipids-phospholipase C, Isc1p [Sawai, H., Okamoto, Y., Luberto, C., Mao, C., Bielawska, A., Domae, M., and Hannun, Y. A. (2000) J. Biol. Chem. 275, 39793-39798]. The present study was conducted to determine specific domains in Isc1p required for catalysis. Several amino acid residues are conserved from bacterial sphingomyelinase to mammalian sphingomyelinase and are also found in ISC1. Individual mutation of the conserved E100, N233, and H334 resulted in complete loss of Isc1p activity, suggesting an essential role in catalysis for these amino acid residues. Isc1p also contains a domain (from G162 to S169) with homology to P-loop domains, found in nucleotide-binding proteins. In addition, two amino acid residues from this domain, D163 and K168, are conserved from bacterial to mammalian sphingomyelinases in this "P-loop-like domain". G162, D163, G167, K168, and S169 were replaced individually with alanine using site-directed mutagenesis. D163A and K168A lost activity completely. Mutations in the other three positions rendered enzyme versions with much reduced but detectable activity. The V(max) values for G162A, G167A, and S169A were reduced, compared with wild type, but the K(m) values for G162A, G167A, and S169A were similar to that of wild type, indicating that the substrate binding efficiency was not greatly altered in these mutants and that the P-loop-like domain of ISC1 might be essential in catalysis of Isc1p. Furthermore, the Mg(2+) K(a) constants for G162A, G167, and S169A were higher than that for wild type, suggesting that this P-loop-like domain may be involved in Mg(2+) binding. Although cell lysates from yeast cells overexpressing all mutants similarly bound to phosphatidylserine (PS), an anionic lipid activator of Isc1p, G162A and G167A required 13.3 mol % PS to achieve maximum activity compared to 6.7 mol % for the wild-type enzyme, suggesting that PS might play a role in optimal catalytic efficiency of Isc1p via this P-loop-like domain. This study provides novel insight into a new domain found in Isc1p and related enzymes.     

Probing the coenzyme specificity of glyceraldehyde-3-phosphate dehydrogenases by site-directed mutagenesis

By combining our knowledge of the crystal structure of the glycolytic NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the sequence of the photosynthetic NADP-dependent GAPDH of the chloroplast, two particular amino acid residues were predicted as the principal determinants of differing coenzyme specificity. By use of site-directed mutagenesis, the amino acids Leu 187 and Pro 188 of GAPDH from Bacillus stearothermophilus have been replaced with Ala 187 and Ser 188, which occur in the sequence from the chloroplast enzyme. The resulting mutant was shown to be catalytically active not only with its natural coenzyme NAD but also with NADP, thus confirming the initial hypothesis. This approach has not only enabled us to alter the coenzyme specificity by minimal amino acid changes but also revealed factors that control the relative affinity of the enzyme for NAD and NADP.     

Reactive-site specificity of human kallistatin toward tissue kallikrein probed by site-directed mutagenesis

Kallistatin is a serine proteinase inhibitor that forms complexes with tissue kallikrein and inhibits its activity. In this study, we compared the inhibitory activity of recombinant human kallistatin and two mutants, Phe388Arg (P1) and Phe387Gly (P2), toward human tissue kallikrein. Recombinant kallistatins were expressed in Escherichia coli and purified to apparent homogeneity using metal-affinity and heparin-affinity chromatography. The complexes formed between recombinant kallistatins and tissue kallikrein were stable for at least 150 h. Wild-type kallistatin as well as both Phe388Arg and Phe387Gly mutants act as inhibitors and substrates to tissue kallikrein as analyzed by complex formation. Kinetic analyses showed that the inhibitory activity of Phe388Arg variant toward tissue kallikrein is two-fold higher than that of wild type (P1Phe), whereas Phe387Gly had only 7% of the inhibitory activity toward tissue kallikrein as compared to wild type. The Phe388Arg variant but not wild type inhibited plasma kallikrein's activity. These results indicate that P1Arg variant exhibits more potent inhibitory activity toward tissue kallikrein while wild type (P1Phe) is a more selective inhibitor of tissue kallikrein. The P2 phenylalanine is essential for retaining the hydrophobic environment for the interaction of kallistatin and kallikrein.     

Probing the specificity of a trypanosomal aromatic alpha-hydroxy acid dehydrogenase by site-directed mutagenesis

The aromatic l-alpha-hydroxy acid dehydrogenase (AHDAH) from Trypanosoma cruzi has over 50% sequence identity with cytosolic malate dehydrogenases (cMDHs), yet it is unable to reduce oxaloacetate. Molecular modeling of the three-dimensional structure of AHADH using the pig cMDH as template directed the construction of several mutants. AHADH shares with MDHs the essential catalytic residues H195 and R171 (using Eventoff's numbering). The AHADH A102R mutant became able to reduce oxaloacetate, while remaining fully active towards aromatic alpha-oxoacids. The Y237G mutant diminished its affinity for all of the natural substrates, whereas the double mutant A102R/Y237G was more active than Y237G and had similar activity with oxaloacetate and with aromatic substrates. The present results reinforce our proposal that AHADH arose by a moderate number of point mutations from a cMDH no longer present in the parasite.     

Modulation of the carbohydrate-binding specificity of two Xenopus proto-type galectins by site-directed mutagenesis

The galectin family is a representative soluble lectin group, which is responsible for the modulation of various cell functions. Although the carbohydrate-binding specificity of galectins has been well-studied, the relationship between protein structure and specificity remains to be elucidated. We previously reported the characteristics of a Xenopus laevis skin galectin, xgalectin-Va, which had diverged from galectin-1. The carbohydrate selectivity of xgalectin-Va was different from that of human galectin-1 and xgalectin-Ib (a Xenopus laevis galectin-1 homolog). In this study, we clarified the key residues for this selectivity by site-directed mutagenesis. Substitution of two amino acids of xgalectin-Va, Val56Gly/Lys76Arg, greatly enhanced the binding ability to N-acetyllactosamine and conferred significant T-cell growth inhibition activity, although the wild type had no activity. These two residues, Gly54 and Arg74 in galectin-1, would cooperatively contribute to the N-acetyllactosamine recognition. The loop region between the S4 and S5 β-strands was involved in the binding to the TF-antigen disaccharide. The loop substitution successfully changed the carbohydrate selectivity of xgalectin-Va and xgalectin-Ib.     

Experimental analysis by site-directed mutagenesis of somatic mutation effects on affinity and fine specificity in antibodies specific for lysozyme.

To experimentally examine the functional roles of somatically derived structural variation in the lysozyme-binding mAb HyHEL-10, we have introduced three different point mutations and one insertion at two different sites in HyHEL-10 by site-directed mutagenesis and expression of the mutant antibodies. Mutation of Asp----Ala at position 101 of the H chain returns a somatically mutated residue to its germline sequence for HyHEL-10, and reduces affinity for chicken lysozyme by approximately 9000-fold. Lengthening the third H chain hypervariable region by two amino acids reduces affinity by about 2000-fold. Two mutations, Asp----Thr at position 101 in the H chain and Lys----Thr at position 49 in the L chain, model somatic differences found in another structurally related but functionally distinguishable mAb and minimally decrease affinity for chicken lysozyme. The H chain mutation Asp101VVH----Thr has little effect on affinity for other avian lysozymes but does alter relative fine specificity for these lysozymes. The L chain mutation Lys49VK----Thr increases affinity for duck lysozyme by approximately fivefold. Neither of the positions mutated, 101 in the H chain nor 49 in the L chain, nor the residues near the insertion contact lysozyme in the x-ray structure of the HyHEL-10 F(ab)-HEL complex. The results suggest that these mutations, which model observed somatic mutations, produce functional variation by indirect or long-range effects.     

Altering the substrate specificity of glutamate dehydrogenase from Bacillus subtilis by site-directed mutagenesis

The Lys80, Gly82 and Met101 residues of glutamate dehydrogenase from Bacillus subtilis were mutated into a series of single mutants. The wild-type enzyme was highly specific for 2-oxoglutarate, whereas G82K and M101S dramatically switched to increased specificity for oxaloacetate with kcat values 3.45 and 5.68 s-1, which were 265-fold and 473-fold higher respectively than those for 2-oxoglutarate.     

Probing the determinants of coenzyme specificity in ferredoxin-NADP+ reductase by site-directed mutagenesis

On the basis of sequence and three-dimensional structure comparison between Anabaena PCC7119 ferredoxin-NADP(+) reductase (FNR) and other reductases from its structurally related family that bind either NADP(+)/H or NAD(+)/H, a set of amino acid residues that might determine the FNR coenzyme specificity can be assigned. These residues include Thr-155, Ser-223, Arg-224, Arg-233 and Tyr-235. Systematic replacement of these amino acids was done to identify which of them are the main determinants of coenzyme specificity. Our data indicate that all of the residues interacting with the 2'-phosphate of NADP(+)/H in Anabaena FNR are not involved to the same extent in determining coenzyme specificity and affinity. Thus, it is found that Ser-223 and Tyr-235 are important for determining NADP(+)/H specificity and orientation with respect to the protein, whereas Arg-224 and Arg-233 provide only secondary interactions in Anabaena FNR. The analysis of the T155G FNR form also indicates that the determinants of coenzyme specificity are not only situated in the 2'-phosphate NADP(+)/H interacting region but that other regions of the protein must be involved. These regions, although not interacting directly with the coenzyme, must produce specific structural arrangements of the backbone chain that determine coenzyme specificity. The loop formed by residues 261-268 in Anabaena FNR must be one of these regions.     

Sterol methyltransferase: functional analysis of highly conserved residues by site-directed mutagenesis

Sterol methyltransferase (SMT), the enzyme from Saccharomyces cerevisiae that catalyzes the conversion of sterol acceptor in the presence of AdoMet to C-24 methylated sterol and AdoHcy, was analyzed for amino acid residues that contribute to C-methylation activity. Site-directed mutagenesis of nine aspartate or glutamate residues and four histidine residues to leucine (amino acids highly conserved in 16 different species) and expression of the resulting mutant proteins in Escherichia coli revealed that residues at H90, Asp125, Asp152, Glu195, and Asp276 are essential for catalytic activity. Each of the catalytically impaired mutants bound sterol, AdoMet, and 25-azalanosterol, a high energy intermediate analogue inhibitor of C-methylation activity. Changes in equilibrium binding and kinetic properties of the mutant enzymes indicated that residues required for catalytic activity are also involved in inhibitor binding. Analysis of the pH dependence of log kcat/Km for the wild-type SMT indicated a pH optimum for activity between 6 and 9. These results and data showing that only the mutant H90L binds sterol, AdoMet, and inhibitor to similar levels as the wild-type enzyme suggest that H90 may act as an acceptor in the coupled methylation-deprotonation reaction. Circular dichroism spectra and chromatographic information of the wild-type and mutant enzymes confirmed retention of the overall conformation of the enzyme during the various experiments. Taken together, our studies suggest that the SMT active center is composed of a set of acidic amino acids at positions 125, 152, 195, and 276, which contribute to initial binding of sterol and AdoMet and that the H90 residue functions subsequently in the reaction progress to promote product formation.     

Analysis of the catalytic specificity of cytochrome P450 enzymes through site-directed mutagenesis.

The way in which structural diversity encodes the capacity of individual P450 enzymes to metabolize multiple, structurally distinct substrates remains largely unknown. The tools of molecular biology provide a means of identifying amino acid residues among closely related P450s that are determinants of their distinct catalytic properties. Work in our laboratory has identified two substrate specificity-determining segments of the amino acid sequences of subfamily 2C P450s. A pattern has emerged from this work, and that of others, which suggests a model for the structural basis of P450 catalytic diversity.     

Investigation of the substrate specificity of a beta-glycosidase from Spodoptera frugiperda using site-directed mutagenesis and bioenergetics analysis.

The specificity of the Spodoptera frugiperda digestive beta-glycosidase (Sfbetagly50) for fucosides, glucosides and galactosides is determined by noncovalent interactions of glycone 6-OH and glycone 4-OH with the active-site residues Q39 and E451. Site-directed mutagenesis and enzyme steady-state kinetics were described, showing that replacement of E451 with glutamine increased the preference of Sfbetagly50 for glucosides in comparison to galactosides, whereas replacing E451 with serine had the opposite effect. In contrast, the replacement of E451 with aspartate did not change Sfbetagly50 specificity. The energy of the interactions formed by these different residues with the axial and equatorial glycone 4-OH were also measured, showing that the increase in preference for galactosides resulted from a larger energy decrease in the interaction with equatorial 4-OH than with axial 4-OH (22.6 vs. 13.9 kJ x mol(-1)), whereas the increase in preference for glucosides was caused by an energy reduction in the interaction with the axial 4-OH (5.1 kJ x mol(-1)). The introduction of glutamine at position 451 or of asparagine at position 39 increased the preference of Sfbetagly50 for fucosides in comparison to galactosides, whereas the presence of aspartate or serine at position 451 had less effect on this preference. The hydrolysis of fucosides was favored because glutamine at position 451 increased a steric hindrance with 6-OH of 7.1 kJ x mol(-1) and asparagine at position 39 disrupted a favorable interaction with this same hydroxyl. In conclusion, it is proposed that the specificity of new beta-glycosidase mutants can be predicted by combining and adding energy of the enzyme-substrate interactions evaluated in the present study.