Structural basis for the substrate specificity of a Bacillus 1,3-1,4-beta-glucanase

Depolymerization of polysaccharides is catalyzed by highly specific enzymes that promote hydrolysis of the scissile glycosidic bond by an activated water molecule. 1,3-1,4-beta-Glucanases selectively cleave beta-1,4 glycosidic bonds in 3-O-substituted glucopyranosyl units within polysaccharides with mixed linkage. The reaction follows a double-displacement mechanism by which the configuration of the anomeric C(1)-atom of the glucosyl unit in subsite -I is retained. Here we report the high-resolution crystal structure of the hybrid 1,3-1,4-beta-glucanase H(A16-M)(E105Q/E109Q) in complex with a beta-glucan tetrasaccharide. The structure shows four beta-d-glucosyl moieties bound to the substrate-binding cleft covering subsites -IV to -I, thus corresponding to the reaction product. The ten active-site residues Asn26, Glu63, Arg65, Phe92, Tyr94, Glu105, Asp107, Glu109, Asn182 and Trp184 form a network of hydrogen bonds and hydrophobic stacking interactions with the substrate. These residues were previously identified by mutational analysis as significant for stabilization of the enzyme-carbohydrate complex, with Glu105 and Glu109 being the catalytic residues. Compared to the Michaelis complex model, the tetrasaccharide moiety is slightly shifted toward that part of the cleft binding the non-reducing end of the substrate, but shows previously unanticipated strong stacking interactions with Phe92 in subsite -I. A number of specific hydrogen-bond contacts between the enzyme and the equatorial O(2), O(3) and O(6) hydroxyl groups of the glucosyl residues in subsites -I, -II and -III are the structural basis for the observed substrate specificity of 1,3-1,4-beta-glucanases. Kinetic analysis of enzyme variants with the all beta-1,3 linked polysaccharide laminarin identified key residues mediating substrate specificity in good agreement with the structural data. The comparison with structures of the apo-enzyme H(A16-M) and a covalent enzyme-inhibitor (E.I) complex, together with kinetic and mutagenesis data, yields new insights into the structural requirements for substrate binding and catalysis. A detailed view of enzyme-carbohydrate interactions is presented and mechanistic implications are discussed.     

Role of hydrogen bond networks and dynamics in positive and negative cooperative stabilization of a protein

Cooperativity mediated through hydrogen bond networks in yeast iso-1-cytochrome c was studied using a thermodynamic triple mutant cycle. Three known stabilizing mutations, Asn 26 to His, Asn 52 to Ile, and Tyr 67 to Phe, were used to construct the triple mutant cycle. The side chain of His 26, a wild-type residue, forms two hydrogen bonds that bridge two substructures of the wild-type protein, and Tyr 67 and Asn 52 are part of an extensive buried hydrogen bond network. The stabilities of all variants in the triple mutant cycle were determined by guanidine hydrochloride denaturation methods and used to determine the pairwise, Delta(2)G(int), and triple interaction energies. His 26 and Ile 52 interact cooperatively (Delta(2)G(int) is 1-2 kcal/mol), whereas the two other pairs of mutations interact anticooperatively (Delta(2)G(int) is -0.5 to -1.5 kcal/mol). Previously reported structural data for iso-1-cytochrome c variants containing these mutations show that changes in the strength of the His 26 to Glu 44 hydrogen bond, apparently caused by changes in main chain dynamics, provide a mechanism for the long distance (His 26 to Phe 67 and His 26 to Ile 52) propagation of pairwise interaction energies. Opposing changes in the strength of the His 26 to Glu 44 hydrogen bond caused by the N52I and Y67F mutations generate a negative triple interaction energy (-0.9 +/-0.7 kcal/mol) that combined with cancellation of cooperative and anticooperative pairwise interactions produce apparent additivity for the stabilizing effects of the single mutations in the triple mutant variant.     

The amino acid sequence of satyr tragopan lysozyme and its activity

The amino acid sequence of satyr tragopan lysozyme and its activity was analyzed. Carboxymethylated lysozyme was digested with trypsin and the resulting peptides were sequenced. The established amino acid sequence had three amino acid substitutions at positions 103 (Asn to Ser), 106 (Ser to Asn), and 121 (His to Gln) comparing with Temminck's tragopan lysozyme and five amino acid substitutions at positions 3 (Phe to Tyr), 15 (His to Leu), 41 (Gln to His), 101 (Asp to Gly) and 103 (Asn to Ser) with chicken lysozyme. The time course analysis using N-acetylglucosamine pentamer as a substrate showed a decrease of binding free energy change, 1.1 kcal/mol at subsite A and 0.2 kcal/mol at subsite B, between satyr tragopan and chicken lysozymes. This was assumed to be responsible for the amino acid substitutions at subsite A-B at position 101 (Asp to Gly), however another substitution at position 103 (Asn to Ser) considered not to affect the change of the substrate binding affinity by the observation of identical time course of satyr tragopan lysozyme with turkey and Temminck's tragopan lysozymes that carried the identical amino acids with chicken lysozyme at this position. These results indicate that the observed decrease of binding free energy change at subsites A-B of satyr tragopan lysozyme was responsible for the amino acid substitution at position 101 (Asp to Gly).     

Amino acid residues in subsites e and f responsible for the characteristic enzymatic activity of duck egg-white lysozyme

We analyzed the enzymatic properties of duck egg-white lysozyme II (DEL), which differs from hen egg-white lysozyme (HEL) in nineteen amino acid substitutions. A substrate binding study showed that DEL binds to the substrate analog at subsites A-C in the same manner as HEL. However, the experimental time-courses of DEL against the substrate N-acetylglucosamine pentamer, (GlcNAc)(5), revealed remarkably enhanced production of (GlcNAc)(2) and reduced production of (GlcNAc)(1) as compared to in the case of HEL. Computer simulation of the DEL-catalyzed reaction suggested that the amino acid substitutions at subsites E and F (Phe34 to Tyr and Asn37 to Ser) caused the great alteration in the time-courses of DEL. Subsequently, the enzymatic reactions of mutants, in which Phe34 and Asn37 in HEL were converted to Tyr and Ser, respectively, were characterized. The time-courses of the F34Y mutant exhibited profiles similar to those of HEL. In contrast, the characteristics of the N37S mutant were different from those of HEL and rather similar to those of DEL; the order of the amounts of (GlcNAc)(1) and (GlcNAc)(2) was reversed in comparison with in the case of HEL. Enhanced production of (GlcNAc)(2) was also observed for the mutant protein, F34Y/N37S, with two substitutions. These results indicated that the substitution of Asn37 with Ser can account, at least in part, for the characteristic time-courses of DEL. Moreover, replacement of Asn37 with Ser reduced the rate constant of transglycosylation. The substitution of the Asn37 residue may affect the transglycosylation activity of HEL.     

alpha-lactalbumin mutant acting as lysozyme

A mutant of alpha-lactalbumin was expressed and purified, in which His32, Thr33, Glu49, Ile59, Val99, and Tyr103 were substituted by Leu32, Glu33, Asp49, Trp59, Asn99, and Ala103, respectively, to create a catalytic site of lysozyme in alpha-lactalbumin. The mutant catalyzed hydrolysis of the synthetic substrate, pNP-(NAcGlc)(3), with a K(M) and k(cat) of 0.160 +/- 0.00986 mmol/L and 3.39 +/- 0. 0456 x10(-5) min(-1), respectively, which was comparable with those of chicken lysozyme of 0.137 +/- 0.0153 mmol/L and 5.25 +/- 0.115 x10(-4) min(-1). By using the Isothermal Titration Calorimetre (ITC), the average binding enthalpy of the mutant or chicken lysozyme with the substrate (chitopentaose) was measured, which was 49.22 KJ/mol for the mutant and 105.47 KJ/mol for chicken lysozyme. In conclusion, the six point mutations occurring in alpha-lactalbumin could be converted into an enzyme that was 17.5-fold less efficient than chicken lysozyme but nevertheless capable of hydrolyzing the glycosidic bond.     

Esterolytic antibodies as mechanistic and structural models of hydrolases-a quantitative analysis

Understanding enzymes quantitatively and mimicking their remarkable catalytic efficiency is a paramount challenge. Here, we applied esterolytic antibodies (the D-Abs) to dissect and quantify individual elements of enzymatic catalysis such as transition state (TS) stabilization, nucleophilic reactivity and conformational changes. Kinetic and mutagenic analysis of the D-Abs were combined with existing structural evidence to show that catalysis by the D-Abs is driven primarily by stabilization of the tetrahedral oxyanionic intermediate of ester hydrolysis formed by the nucleophilic attack of an exogenous (solution) hydroxide anion. The side-chain of TyrH100d is shown to be the main H-bond donor of the D-Abs oxyanion hole. The pH-rate and pH-binding profiles indicate that the strength of this H-bond increases dramatically as the neutral substrate develops into the oxyanionic TS, resulting in TS stabilization of 5-7 kcal/mol, which is comparable to oxyanionic TS stabilization in serine hydrolases. We show that the rate of the exogenous (intermolecular) nucleophilic attack can be enhanced by 2000-fold by replacing the hydroxide nucleophile with peroxide, an alpha-nucleophile that is much more reactive than hydroxide. In the presence of peroxide, the rate saturates (k(cat)(max)) at 6 s(-1). This rate-ceiling appears to be dictated by the rate of the induced-fit conformational rearrangement leading to the active antibody-TS complex. The selective usage of negatively charged exogenous nucleophiles by the D-Abs led to the identification of a positively charged channel. Imprinted by the negatively-charged TS-analogue against which these antibodies were elicited, this channel presumably directs the nucleophile to the antibody-bound substrate. Our findings are discussed in comparison with serine esterases and, in particular, with cocaine esterase (cocE), which possesses a tyrosine based oxyanion hole.     

Energetic and mechanistic studies of glucoamylase using molecular recognition of maltose OH groups coupled with site-directed mutagenesis

Molecular recognition using a series of deoxygenated maltose analogues was used to determine the substrate transition-state binding energy profiles of 10 single-residue mutants at the active site of glucoamylase from Aspergillus niger. The individual contribution of each substrate hydroxyl group to transition-state stabilization with the wild type and each mutant GA was determined from the relation Delta(DeltaG()) = -RT ln[(k(cat)/K(M))(x)/(k(cat)/K(M))(y)], where x represents either a mutant enzyme or substrate analogue and y the wild-type enzyme or parent substrate. The resulting binding energy profiles indicate that disrupting an active site hydrogen bond between enzyme and substrate, as identified in crystal structures, not only sharply reduces or eliminates the energy contributed from that particular hydrogen bond but also perturbs binding contributions from other substrate hydroxyl groups. Replacing the active site acidic groups, Asp55, Glu180, or Asp309, with the corresponding amides, and the neutral Trp178 with the basic Arg, all substantially reduced the binding energy contribution of the 4'- and 6'-OH groups of maltose at subsite -1, even though both Glu180 and Asp309 are localized at subsite 1. In contrast, the substitution, Asp176 --> Asn, located near subsites -1 and 1, did not substantially perturb any of the individual hydroxyl group binding energies. Similarly, the substitutions Tyr116 --> Ala, Ser119 --> Tyr, or Trp120 --> Phe also did not substantially alter the energy profiles even though Trp120 has a critical role in directing conformational changes necessary for activity. Since the mutations at Trp120 and Asp176 reduced k(cat) values by 50- and 12-fold, respectively, a large effect on k(cat) is not necessarily accompanied by changes in hydroxyl group binding energy contributions. Two substitutions, Asn182 --> Ala and Tyr306 --> Phe, had significant though small effects on interactions with 3- and 4'-OH, respectively. Binding interactions between the enzyme and the glucosyl group in subsite -1, particularly with the 4'- and 6'-OH groups, play an important role in substrate binding, while subsite 1 interactions may play a more important role in product release.     

Chitin oligosaccharide binding to a family GH19 chitinase from the moss Bryum coronatum

Substrate binding of a family GH19 chitinase from a moss species, Bryum coronatum (BcChi-A, 22 kDa), which is smaller than the 26 kDa family GH19 barley chitinase due to the lack of several loop regions ('loopless'), was investigated by oligosaccharide digestion, thermal unfolding experiments and isothermal titration calorimetry (ITC). Chitin oligosaccharides [β-1,4-linked oligosaccharides of N-acetylglucosamine with a polymerization degree of n, (GlcNAc)(n), n = 3-6] were hydrolyzed by BcChi-A at rates in the order (GlcNAc)(6) > (GlcNAc)(5) > (GlcNAc)(4) > (GlcNAc)(3). From thermal unfolding experiments using the inactive BcChi-A mutant (BcChi-A-E61A), in which the catalytic residue Glu61 is mutated to Ala, we found that the transition temperature (T(m) ) was elevated upon addition of (GlcNAc)(n) (n = 2-6) and that the elevation (ΔT(m)) was almost proportional to the degree of polymerization of (GlcNAc)(n). ITC experiments provided the thermodynamic parameters for binding of (GlcNAc)(n) (n = 3-6) to BcChi-A-E61A, and revealed that the binding was driven by favorable enthalpy changes with unfavorable entropy changes. The change in heat capacity (ΔC(p)°) for (GlcNAc)(6) binding was found to be relatively small (-105 ± 8 cal·K(-1) ·mol(-1)). The binding free energy changes for (GlcNAc)(6), (GlcNAc)(5), (GlcNAc)(4) and (GlcNAc)(3) were determined to be -8.5, -7.9, -6.6 and -5.0 kcal·mol(-1), respectively. Taken together, the substrate binding cleft of BcChi-A consists of at least six subsites, in contrast to the four-subsites binding cleft of the 'loopless' family 19 chitinase from Streptomyces coelicolor. DATABASE: Chitinase, EC 3.2.1.14.     

The cystine knot promotes folding and not thermodynamic stability in vascular endothelial growth factor

Cystine knots consist of three intertwined disulfide bridges and are considered major determinants of protein stability in proteins in which they occur. We questioned this function and observed that removal of individual disulfide bridges in human vascular endothelial growth factor (VEGF) does not reduce its thermodynamic stability but reduces its unexpected high thermal stability of 108 degrees C by up to 40 degrees C. In wild-type VEGF (deltaG(u,25)(0) = 5.1 kcal.mol(-1)), the knot is responsible for a large entropic stabilization of TdeltaS(u,25)(0) = -39.3 kcal mol(-1), which is compensated for by a deltaH(u,25)(0) of -34.2 kcal mol(-1). In the disulfide-deficient mutants, this entropic stabilization disappears, but instead of a decrease, we observe an increase in the thermodynamic stability by about 2 kcal.mol(-1). A detailed crystallographic analysis of the mutant structures suggests a role of the cystine knot motif in protein folding rather than in the stabilization of the folded state. When assuming that the sequential order of the disulfide bridge formation is conserved between VEGF and glycoprotein alpha-subunit, the crystal structure of the mutant C61A-C104A, which deviates by a root mean square deviation of more than 2.2 A from wild-type VEGF, identifies a true folding intermediate of VEGF.     

X-ray structure of papaya chitinase reveals the substrate binding mode of glycosyl hydrolase family 19 chitinases

The crystal structure of a chitinase from Carica papaya has been solved by the molecular replacement method and is reported to a resolution of 1.5 A. This enzyme belongs to family 19 of the glycosyl hydrolases. Crystals have been obtained in the presence of N-acetyl- d-glucosamine (GlcNAc) in the crystallization solution and two well-defined GlcNAc molecules have been identified in the catalytic cleft of the enzyme, at subsites -2 and +1. These GlcNAc moieties bind to the protein via an extensive network of interactions which also involves many hydrogen bonds mediated by water molecules, underlying their role in the catalytic mechanism. A complex of the enzyme with a tetra-GlcNAc molecule has been elaborated, using the experimental interactions observed for the bound GlcNAc saccharides. This model allows to define four major substrate interacting regions in the enzyme, comprising residues located around the catalytic Glu67 (His66 and Thr69), the short segment E89-R90 containing the second catalytic residue Glu89, the region 120-124 (residues Ser120, Trp121, Tyr123, and Asn124), and the alpha-helical segment 198-202 (residues Ile198, Asn199, Gly201, and Leu202). Water molecules from the crystal structure were introduced during the modeling procedure, allowing to pinpoint several additional residues involved in ligand binding that were not previously reported in studies of poly-GlcNAc/family 19 chitinase complexes. This work underlines the role played by water-mediated hydrogen bonding in substrate binding as well as in the catalytic mechanism of the GH family 19 chitinases. Finally, a new sequence motif for family 19 chitinases has been identified between residues Tyr111 and Tyr125.     

Structural rationale for low-nanomolar binding of transition state mimics to a family GH3 beta-D-glucan glucohydrolase from barley

The interactions of a transition state mimic anilinomethyl glucoimidazole (AmGlcIm), with a K(i) constant of 0.6 x 10(-)(9) M and a Gibbs free energy value of -53.5 kJ/mol, with a family GH3 beta-d-glucan glucohydrolase from barley have been analyzed crystallographically and by ab initio quantum mechanical modeling. AmGlcIm binds 3 times more tightly to the beta-d-glucan glucohydrolase than a previously investigated phenyl glucoimidazole. In the enzyme-AmGlcIm complex, an additional residue, Tyr253, and a water molecule positioned between subsites -1 and +1 are recruited for binding. Analyses of the two binary complexes reveal the following. (i) An intricate network exists in which hydrogen bonds between the enzyme's catalytic pocket residues Lys206, His207, Tyr253, Asp285, and Glu491 and the glucoimidazoles are shorter by 0.15-0.53 A, compared with distances of hydrogen bonds in the Michaelis complex. (ii) The "glucose" moiety of the glucoimidazoles adopts a (4)E conformation that is vital for the low-nanomolar binding. (iii) The N1 atoms of the glucoimidazoles are positioned nearly optimally for in-line protonation by the Oepsilon1 atom of the catalytic acid/base Glu491. (iv) The enzyme derives binding energies from both glycone and aglycone components of the glucoimidazoles. (iv) The prevalent libration motion of the two domains of the enzyme could play a significant role during induced fit closure in the active site. (v) Modeling based on the structural data predicts that protons could be positioned on the N1 atoms of the glucoimidazoles, and the catalytic acid/base Glu491 could carry an overall negative charge. (vi) The enzyme-AmGlcIm complex reveals the likely structure of an early transition state during hydrolysis. Finally, the high-resolution structures enabled us to define minimal structures of oligosaccharides attached to Asn221, Asn498, and Asn600 N-glycosylation sites.     

The Amino Acid Sequence of Monal Pheasant Lysozyme and Its Activity

The amino acid sequence of monal pheasant lysozyme and its activity were analyzed. Carboxymethylated lysozyme was digested with trypsin and the resulting peptides were sequenced. The established amino acid sequence had one amino acid substitution at position 102 (Arg to Gly) comparing with Indian peafowl lysozyme and four amino acid substitutions at positions 3 (Phe to Tyr), 15 (His to Leu), 41 (Gln to His), and 121 (Gln to His) with chicken lysozyme. Analysis of the time-courses of reaction using N-acetylglucosamine pentamer as a substrate showed a difference of binding free energy change (-0.4 kcal/mol) at subsites A between monal pheasant and Indian peafowl lysozyme. This was assumed to be caused by the amino acid substitution at subsite A with loss of a positive charge at position 102 (Arg102 to Gly).     

Effects of structure on alpha C-H bond enthalpies of amino acid residues: relevance to H transfers in enzyme mechanisms and in protein oxidation.

The bond dissociation enthalpies (BDE) of all of the amino acid residues, modeled by HC(O)NHCH(R)C(O)NH(2) (PH(res)), were determined at the B3LYP/6-31G//B3LYP/6-31G level, coupled with isodesmic reactions. The results for neutral side chains with phi, psi angles approximately 180 degrees, approximately 180 degrees in ascending order, to an expected accuracy of +/-10 kJ mol(-)(1), are Asn 326; cystine 330; Asp 332; Gln 334; Trp 337; Arg 340; Lys 340; Met 343; His 344; Phe 344; Tyr 344; Leu 344; Ala 345; Cys 346; Ser 349; Gly 350; Ile 351; Val 352; Glu 354; Thr 357; Pro-cis 358; Pro-trans 369. BDEs calculated at the ROMP2/6-31G//B3LYP/6-31G level exhibit the same trends but are approximately 7 kJ mol(-)(1) higher. All BDEs are smaller than those of typical secondary or tertiary C-H bonds due to the phenomenon of captodative stabilization. The stabilization is reduced by changes in the phi,psi angles. As a result the BDEs increase by about 10 kJ mol(-)(1) in beta-sheet and 40 kJ mol(-)(1) in alpha-helical environments, respectively. In effect the alpha C-H BDEs can be "tuned" from about 345 to 400 kJ mol(-)(1) by adjusting the local environment. Some very significant effects of this are seen in the current literature on H-transfer processes in enzyme mechanisms and in oxidative damage to proteins. These observations are discussed in terms of the findings of the present study.     

Contribution of surface salt bridges to protein stability

The role of surface salt bridges in protein stabilization has been a source of controversy. Here we present the NMR structure of a hyperthermophilic rubredoxin variant (PFRD-XC4) and the thermodynamic analysis of two surface salt bridges by double mutant cycles. This analysis shows that the surface side chain to side chain salt bridge between Lys 6 and Glu 49 does not stabilize PFRD-XC4. The main chain to side chain salt bridge between the N-terminus and Glu 14 was, however, found to stabilize PFRD-XC4 by 1. 5 kcal mol(-)(1). The entropic cost of making a surface salt bridge involving the protein's backbone is reduced, since the backbone has already been immobilized upon protein folding.     

The active site of an acidic endo-1,4-beta-xylanase of Aspergillus niger

The substrate binding site of an acidic endo-1,4-beta-xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) of Aspergillus niger was investigated using 1,4-beta-xylooligosaccharides (1-3H)-labelled at the reducing end. Bond cleavage frequencies and V/Km parameters of the oligosaccharides were determined under conditions of unimolecular hydrolysis and, according to the method of Suganuma et al. (J. Biochem. (Tokyo) (1978) 84, 293-316), used for evaluation of subsite affinities. The substrate binding site of the enzyme was found to consist of seven subsites, numbered -IV, -III, -II, -I, I, II and III, towards the subsite binding the reducing end unit of xyloheptaose. The catalytic groups were localized between subsites -I and I, the affinities of which have not been determined. All other subsites showed positive values of affinities for binding xylosyl residues. The values decrease from subsites -II and II, similarly in both directions. As a consequence of such an almost symmetric distribution of affinities around the catalytic groups, the enzyme cleaves preferentially the bonds in the oligosaccharides which are most distant from both terminals. Thus, the acidic A. niger beta-xylanase appears to be an endo-1,4-beta-xylanase attacking polymeric substrates in a random fashion. This conclusion was supported by viscosimetric measurements with carboxymethylxylan as a substrate.     

Functional and structural effects of mutagenic replacement of Asn37 at subsite F on the lysozyme-catalyzed reaction

To investigate the functional role of subsites E and F in lysozyme catalysis, Asn37 of hen egg-white lysozyme (HEL), which is postulated to participate in sugar residue binding at the right-sided subsite F through hydrogen bonding, was replaced by Ser or Gly by site-directed mutagenesis. The mutations of Asn37 neither significantly affected the binding constant for chitotriose nor the enzymatic activity toward the substrate glycol chitin. However, kinetic analysis with the substrate N-acetylglucosamine pentamer, (GlcNAc)(5), revealed that the conversion of Asn37 to Gly decreased the binding free energies for subsites E and F, while the conversion to Ser increased the substrate affinity at subsite F. It was further found that the rate constant of transglycosylation was reduced by these mutations. These results suggest that Asn37 is involved not only in substrate binding at subsite F but also in transglycosylation activity. No remarkable change in the tertiary structure except the side chain of the 37th residue was detected on X-ray analysis of the mutant proteins, indicating that the alterations in the enzymatic function between the wild type and mutant enzymes depend on limited structural change around the substitution site. It is thus speculated that the slight conformational difference in the side chain of position 37 may affect the substrate and acceptor binding at subsites E and F, leading to lower the efficiency of the transglycosylation activities of the mutant proteins.     

Expression and characterization of active site mutants of hevamine, a chitinase from the rubber tree Hevea brasiliensis.

Hevamine is a chitinase from the rubber tree Hevea brasiliensis. Its active site contains Asp125, Glu127, and Tyr183, which interact with the -1 sugar residue of the substrate. To investigate their role in catalysis, we have successfully expressed wild-type enzyme and mutants of these residues as inclusion bodies in Escherichia coli. After refolding and purification they were characterized by both structural and enzyme kinetic studies. Mutation of Tyr183 to phenylalanine produced an enzyme with a lower k(cat) and a slightly higher K(m) than the wild-type enzyme. Mutating Asp125 and Glu127 to alanine gave mutants with approximately 2% residual activity. In contrast, the Asp125Asn mutant retained substantial activity, with an approximately twofold lower k(cat) and an approximately twofold higher K(m) than the wild-type enzyme. More interestingly, it showed activity to higher pH values than the other variants. The X-ray structure of the Asp125Ala/Glu127Ala double mutant soaked with chitotetraose shows that, compared with wild-type hevamine, the carbonyl oxygen atom of the N-acetyl group of the -1 sugar residue has rotated away from the C1 atom of that residue. The combined structural and kinetic data show that Asp125 and Tyr183 contribute to catalysis by positioning the carbonyl oxygen of the N-acetyl group near to the C1 atom. This allows the stabilization of a positively charged transient intermediate, in agreement with a previous proposal that the enzyme makes use of substrate-assisted catalysis.     

Active site of chondroitin AC lyase revealed by the structure of enzyme-oligosaccharide complexes and mutagenesis

The crystal structures of Flavobacterium heparinium chondroitin AC lyase (chondroitinase AC; EC 4.2.2.5) bound to dermatan sulfate hexasaccharide (DS(hexa)), tetrasaccharide (DS(tetra)), and hyaluronic acid tetrasaccharide (HA(tetra)) have been refined at 2.0, 2.0, and 2.1 A resolution, respectively. The structure of the Tyr234Phe mutant of AC lyase bound to a chondroitin sulfate tetrasaccharide (CS(tetra)) has also been determined to 2.3 A resolution. For each of these complexes, four (DS(hexa) and CS(tetra)) or two (DS(tetra) and HA(tetra)) ordered sugars are visible in electron density maps. The lyase AC DS(hexa) and CS(tetra) complexes reveal binding at four subsites, -2, -1, +1, and +2, within a narrow and shallow protein channel. We suggest that subsites -2 and -1 together represent the substrate recognition area, +1 is the catalytic subsite and +1 and +2 together represent the product release area. The putative catalytic site is located between the substrate recognition area and the product release area, carrying out catalysis at the +1 subsite. Four residues near the catalytic site, His225, Tyr234, Arg288, and Glu371 together form a catalytic tetrad. The mutations His225Ala, Tyr234Phe, Arg288Ala, and Arg292Ala, revealed residual activity for only the Arg292Ala mutant. Structural data indicate that Arg292 is primarily involved in recognition of the N-acetyl and sulfate moieties of galactosamine, but does not participate directly in catalysis. Candidates for the general base, removing the proton attached to C-5 of the glucuronic acid at the +1 subsite, are Tyr234, which could be transiently deprotonated during catalysis, or His225. Tyrosine 234 is a candidate to protonate the leaving group. Arginine 288 likely contributes to charge neutralization and stabilization of the enolate anion intermediate during catalysis.     

Tyrosine hydrogen-bonding and environmental effects in proteins probed by ultraviolet resonance Raman spectroscopy

Ultraviolet resonance Raman spectra with 229-nm excitation are reported for aqueous tyrosine and for ovomucoid third domain proteins from chicken [OMCHI3(-)] and from chachalaca [OMCHA(-)], as well as alpha 1-, alpha 2-, and beta-purothionin. At this excitation wavelength interference from phenylalanine is minimized, and it is possible to determine the frequencies of the Tyr ring modes nu 8a and nu 8b. The nu 8b frequency decreases with the degree of Tyr H-bond donation, reaching a limiting value for deprotonated tyrosine. This spectroscopic indicator of H-bond strength was calibrated by using the model compound p-cresol in H-bond acceptor solutions for which the enthalpy of H-bond formation can be obtained from the literature. With this calibration it is possible to estimate Tyr H-bond enthalpies in proteins for which Tyr is a H-bond donor; values of 13.7, 9.6, and 11.2 kcal/mol were found for OMCHA3(-) and for alpha 1- (or alpha 2-) and beta-purothionin, respectively. The intensity of the 1176-cm-1 nu 9a band of Tyr excited at 229 nm and also the intensity ratio of the Tyr 830/850-cm-1 Fermi doublet excited at 200 nm both correlate strongly with the estimated H-bond enthalpies, but large deviations are seen for the purothionins, reflecting a special environment for the Tyr residue of these proteins, which is believed to be constrained in a hydrophobic pocket. The molar intensity of the strong approximately 1000-cm-1 nu 12 band of phenylalanine in aqueous solution is about half the value observed in most proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rational design of a lipase to accommodate catalysis of Baeyer–Villiger oxidation with hydrogen peroxide

The mechanism and potential energy surface for the Baeyer-Villiger oxidation of acetone with hydrogen peroxide catalyzed by a Ser105-Ala mutant of Candida antarctica Lipase B has been determined using ab initio and density functional theories. Initial substrate binding has been studied using an automated docking procedure and molecular dynamics simulations. Substrates were found to bind to the active site of the mutant. The activation energy for the first step of the reaction, the nucleophilic attack of hydrogen peroxide on the carbonyl carbon of hydrogen peroxide, was calculated to be 4.4 kcal x mol(-1) at the B3LYP/6-31+G* level. The second step, involving the migration of the alkyl group, was found to be the rate-determining step with a computed activation energy of 19.9 kcal x mol(-1) relative the reactant complex. Both steps were found to be lowered considerably in the reaction catalyzed by the mutated lipase, compared to the uncatalyzed reaction. The first step was lowered by 36.0 kcal x mol(-1) and the second step by 19.5 kcal x mol(-1). The second step of the reaction, the rearrangement step, has a high barrier of 27.7 kcal x mol(-1) relative to the Criegee intermediate. This could lead to an accumulation of the intermediate. It is not clear whether this result is an artifact of the computational procedure, or an indication that further mutations of the active site are required. Figure Second TS (18TS) in the Baeyer-Villiger oxidation in a mutant of CALB. Distances in A