Probing the mechanism and improving the rate of substrate-assisted catalysis in subtilisin BPN'

A mutant of the serine protease, subtilisin BPN', in which the catalytic His64 is replaced by Ala (H64A), is very specific for substrates containing a histidine, presumably by the substrate-bound histidine assisting in catalysis [Carter, P., & Wells, J.A. (1987) Science (Washington, D.C.) 237, 394-399]. Here we probe the catalytic mechanism of H64A subtilisin for cleaving His and non-His substrates. We show that the ratio of aminolysis to hydrolysis is the same for ester and amide substrates as catalyzed by the H64A subtilisin. This is consistent with formation of a common acyl-enzyme intermediate for H64A subtilisin, analogous to the mechanism of the wild-type enzyme. However, the catalytic efficiencies (kcat/KM) for amidase and esterase activities with His-containing substrates are reduced by 5000-fold and 14-fold, respectively, relative to wild-type subtilisin BPN, suggesting that acylation is more compromised than deacylation in the H64A mutant. High concentrations of imidazole are much less effective than His substrates in promoting hydrolysis by the H64A variant, suggesting that the His residue on the bound (not free) substrate is involved in catalysis. The reduction in catalytic efficiency kcat/KM for hydrolysis of the amide substrate upon replacement of the oxyanion stabilizing asparagine (N155G) is only 7-fold greater for wild-type than H64A subtilisin. In contrast, the reductions in kcat/KM upon replacement of the catalytic serine (S221A) or aspartate (D32A) are about 3000-fold greater for wild-type than H64A subtilisin, suggesting that the functional interactions between the Asp32 and Ser221 with the substrate histidine are more compromised in substrate-assisted catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Semiquantitative calculations of catalytic free energies in genetically modified enzymes

The catalytic free energy and binding free energies of the native and the Asn-155----Thr, Asn-155----Leu, and Asn-155----Ala mutants of subtilisin are calculated by the empirical valence bond method and a free energy perturbation method. Two simple procedures are used; one "mutates" the substrate, and the other "mutates" the enzyme. The calculated changes in free energies (delta delta G not equal to cat and delta delta Gbind) between the mutant and native enzymes are within 1 kcal/mol of the corresponding observed values. This indicates that we are approaching a quantitative structure-function correlation. The calculated changes in catalytic free energies are almost entirely due to the electrostatic interaction between the enzyme-water system and the charges of the reacting system. This supports the idea that the electrostatic free energy associated with the changes of charges of the reacting system is the key factor in enzyme catalysis.     

The importance of a distal hydrogen bonding group in stabilizing the transition state in subtilisin BPN'

Stabilization of an oxyanion transition state is important to catalysis of peptide bond hydrolysis in all proteases. For subtilisin BPN', a bacterial serine protease, structural data suggest that two hydrogen bonds stabilize the tetrahedral-like oxyanion intermediate: one from the main chain NH of Ser221 and another from the side chain NH2 of Asn155. Molecular dynamic studies (Rao, S., N., Singh, U., C. Bush, P. A., and Kollman, P. A. (1987) Nature 328, 551-554) have indicated the gamma-hydroxyl of Thr220 may be a third hydrogen bond donor even though it is 4A away in the static x-ray structure. We have probed the role of Thr220 by replacing it with serine, cysteine, valine, or alanine by site-directed mutagenesis. These substitutions were intended to alter the size and hydrogen bonding ability of residue 220. Removal of the gamma-hydroxyl group reduced the transition state stabilization energy (delta delta GT) by 1.8-2.1 kcal/mol depending upon the substitution. By comparison, removal of the gamma-methyl group in the Thr220 to serine mutation only decreased delta GT by 0.5 kcal/mol. The gamma-hydroxyl of Thr220 is most important for catalysis, not substrate binding, because virtually all of the effects were on kcat and not KM. The role of the Thr220 hydroxyl is functionally independent from the amide NH2 of Asn155 because the free energy effects of double alanine mutants at these two positions are additive. These data indicate that a distal hydrogen bond donor, namely the hydroxyl of Thr220, plays a functionally important role in stabilizing the oxyanion transition state in subtilisin which is independent of Asn155.     

Evidence for transition-state stabilization by serine-148 in the catalytic mechanism of chloramphenicol acetyltransferase

The function of conserved Ser-148 of chloramphenicol acetyltransferase (CAT) has been investigated by site-directed mutagenesis. Modeling studies (P. C. E. Moody and A. G. W. Leslie, unpublished results) suggested that the hydroxyl group of Ser-148 could be involved in transition-state stabilization via a hydrogen bond to the oxyanion of the putative tetrahedral intermediate. Replacement of serine by alanine results in a mutant enzyme (Ala-148 CAT) with kcat reduced 53-fold and only minor changes in Km values for chloramphenicol and acetyl-CoA. The Ser-148----Gly substitution gives rise to a mutant enzyme (Gly-148 CAT) with kcat reduced only 10-fold. A water molecule may partially replace the hydrogen-bonding potential of Ser-148 in Gly-148 CAT. The three-dimensional structure of Ala-148 CAT at 2.34-A resolution is isosteric with that of wild-type CAT with two exceptions: the absence of the Ser-148 hydroxyl group and the loss of one poorly ordered water molecule from the active site region. The results are consistent with a catalytic role for Ser-148 rather than a structural one and support the hypothesis that Ser-148 is involved in transition-state stabilization. Ser-148 has also been replaced with cysteine and asparagine; the Ser-148----Cys mutation results in a 705-fold decrease in kcat and the Ser-148----Asn substitution in a 214-fold reduction in kcat. Removing the hydrogen bond donor (Ser-148----Ala or Gly) is less deleterious than replacing Ser-148 with alternative possible hydrogen bond donors (Ser-148----Cys or Asn).     

X-ray crystallographic study of boronic acid adducts with subtilisin BPN' (Novo). A model for the catalytic transition state.

We have studied the structures of adducts formed between subtilisin BPN' and both benzeneboronic acid and 2-phenylethaneboronic acid by x-ray diffraction techniques. Electron density and difference maps at 2.5 A resolution were computed with phases calculated from a partially refined structure of the native enzyme (R = 0.23 at 2.0 A). Both adducts contain a covalent bond between Ogamma of the catalytic Ser-221 and the inhibitor boron atom. The boron atom is coordinated tetrahedrally, with one of the two additional boronic acid oxygen atoms lying in the "oxyanion hole" and the other at the leaving group site identified in previous studies (ROBERTUS, J.D., Kraut, J. ALDEN, R.A., and BIRKTOFT, J.J. (1972) Biochemistry 11, 4293-4303). Moreover, the previously postulated structure of the tetrahedral intermediate for substrate hydrolysis is isosteric with these boronic acid adducts, which can therefore be considered good models for the transition state complex (KOEHLER, K.K., and LIENHARD, G.E. (1972) Biochemistry 10, 2477-2483). These observations further support the suggestion that an important contribution to stabilization of this transition state complex, relative to both the Michaelis complex and the acyl intermediate, occurs as a consequence of hydrogen bond donation to the substrate carbonyl oxygen atom from the side chain amido group of Asn-155 and from the backbone amido group of Ser-221.     

Are turns required for the folding of ribonuclease T1?

Ribonuclease T1 (RNase T1) is a small, globular protein of 104 amino acids for which extensive thermodynamic and structural information is known. To assess the specific influence of variations in amino acid sequence on the mechanism for protein folding, circularly permuted variants of RNase T1 were constructed and characterized in terms of catalytic activity and thermodynamic stability. The disulfide bond connecting Cys-2 and Cys-10 was removed by mutation of these residues to alanine (C2, 10A) to avoid potential steric problems imposed by the circular permutations. The original amino-terminus and carboxyl-terminus of the mutant (C2, 10A) were subsequently joined with a tripeptide linker to accommodate a reverse turn and new termini were introduced throughout the primary sequence in regions of solvent-exposed loops at Ser-35 (cp35S1), Asp-49 (cp49D1), Gly-70 (cp70G1), and Ser-96 (cp96S1). These circularly permuted RNase T1 mutants retained 35-100% of the original catalytic activity for the hydrolysis of guanylyl(3'-->5')cytidine, suggesting that the overall tertiary fold of these mutants is very similar to that of wild-type protein. Chemical denaturation curves indicated thermodynamic stabilities at pH 5.0 of 5.7, 2.9, 2.6, and 4.6 kcal/mol for cp35S1, cp49D1, cp70G1, and cp96S1, respectively, compared to a value of 10.1 kcal/mol for wild-type RNase T1 and 6.4 kcal/mol for (C2, 10A) T1. A fifth set of circularly permuted variants was attempted with new termini positioned in a tight beta-turn between Glu-82 and Gln-85. New termini were inserted at Asn-83 (cp83N1), Asn-84 (cp84N1), and Gln-85 (cp85Q1). No detectable amount of protein was ever produced for any of the mutations in this region, suggesting that this turn may be critical for the proper folding and/or thermodynamic stability of RNase T1.     

Contribution of peptide bonds to inhibitor-protease binding: crystal structures of the turkey ovomucoid third domain backbone variants OMTKY3-Pro18I and OMTKY3-psi[COO]-Leu18I in complex with Streptomyces griseus proteinase B (SGPB) and the structure of the free inhibitor, OMTKY-3-psi[CH2NH2+]-Asp19I

X-ray crystallography has been used to determine the 3D structures of two complexes between Streptomyces griseus proteinase B (SGPB), a bacterial serine proteinase, and backbone variants of turkey ovomucoid third domain (OMTKY3). The natural P1 residue (Leu18I) has been substituted by a proline residue (OMTKY3-Pro18I) and in the second variant, the peptide bond between Thr17I and Leu18I was replaced by an ester bond (OMTKY3-psi[COO]-Leu18I). Both variants lack the P1 NH group that donates a bifurcated hydrogen bond to the carbonyl O of Ser214 and O(gamma) of the catalytic Ser195, one of the common interactions between serine proteinases and their canonical inhibitors. The SGPB:OMTKY3-Pro18I complex has many structural differences in the vicinity of the S1 pocket when compared with the previously determined structure of SGPB:OMTKY3-Leu18I. The result is a huge difference in the DeltaG degrees of binding (8.3 kcal/mol), only part of which can be attributed to the missing hydrogen bond. In contrast, very little structural difference exists between the complexes of SGPB:OMTKY3-psi[COO]-Leu18I and SGPB:OMTKY3-Leu18I, aside from an ester O replacing the P1 NH group. Therefore, the difference in DeltaG degrees, 1.5 kcal/mol as calculated from the measured equilibrium association constants, can be attributed to the contribution of the P1 NH hydrogen bond toward binding. A crystal structure of OMTKY3 having a reduced peptide bond between P1 Leu18I and P'1 Asp19I, (OMTKY3-psi[CH2NH2+]-Asp19I) has also been determined by X-ray crystallography. This variant has very weak association equilibrium constants with SGPB and with chymotrypsin. The structure of the free inhibitor suggests that the reduced peptide bond has not introduced any major structural changes in the inhibitor. Therefore, its poor ability to inhibit serine proteinases is likely due to the disruptions of the canonical interactions at the oxyanion hole.     

Catalytic and conformational changes induced by limited subtilisin cleavage of bovine carboxypeptidase A.

Limited proteolysis of carboxypeptidase A from bovine pancreas with subtilisin Carlsberg generates a stable intermediate, carboxypeptidase S, whose esterase and peptidase activities are increased and decreased, respectively, under standard assay conditions. Carboxypeptidase S was isolated by affinity chromatography. Sequence analysis shows that it is cleaved solely at the Ala154-Gly155 bond. Its enzymatic properties were determined under stopped-flow conditions with Dns-Gly-Ala-Phe and its ester analogue Dns-Gly-Ala-OPhe. For both substrates, the Km values are increased 30-40-fold. The kcat value for peptide hydrolysis is virtually unaffected whereas that for ester hydrolysis is increased 10-fold. The magnitude of the Km effect is equivalent to a loss of 9 kJ/mol of binding energy and likely reflects a disruption of the network of hydrogen bonds that links Tyr-248 and Arg-145 to the backbone carbonyls of Ala-154 and Gly-155. The difference in kcat effects for the two substrate classes is related to differences in the chemical nature of the rate-determining step. Product release is rate determining for catalytic hydrolysis of ester substrates, and hence, the increase in kcat indicates that dissociation of products is facilitated as a result of the Ala154-Gly155 bond scission. The changes in enzymatic activity accompanying limited proteolysis are due to conformational alterations in the vicinity of the active center of the molecule. The affinity of a monoclonal antibody, mAb 100, directed toward the antigenic determinant located between residues 209 and 218 in carboxypeptidase A is diminished considerably for carboxypeptidase S.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamics and kinetics of the hydrolysis and resynthesis of the reactive site peptide bond in turkey ovomucoid third domain by aspergillopeptidase B

1. Aspergillopeptidase B rapidly hydrolyses the -Leu18-Glu19-reactive site peptide bond in turkey ovomucoid third domain (OMTKY3) within the pH-range of 4.0-8.4. The reaction proceeds to equilibrium between OMTKY3 and its modified form with the reactive site peptide bond cleaved (OMTKY3). 2. The dependence of the equilibrium constant (Khyd) on pH indicates that hydrolysis of the reactive site peptide bond apparently does not perturb the pK-values of any preexistent ionizable groups in OMTKY3. 3. The obtained Khyd0 value indicates that free energies of OMTKY3 and OMTKY3 are essentially the same. 4. Hydrolysis of the reactive site peptide bond by aspergillopeptidase B at neutral pH is about 60 times faster than the same reaction catalyzed by subtilisin (Carlsberg), the enzyme strongly inhibited by OMTKY3. 5. Resynthesis of the reactive site peptide bond at neutral pH catalyzed by aspergillopeptidase B (reverse reaction) is almost four orders of magnitude faster than the forward reaction.     

Kinetics and thermodynamics of mandelate racemase catalysis

Mandelate racemase (EC 5.1.2.2) from Pseudomonas putida catalyzes the interconversion of the two enantiomers of mandelic acid with remarkable proficiency, producing a rate enhancement exceeding 15 orders of magnitude. The rates of the forward and reverse reactions catalyzed by the wild-type enzyme and by a sluggish mutant (N197A) have been studied in the absence and presence of several viscosogenic agents. A partial dependence on relative solvent viscosity was observed for values of kcat and kcat/Km for the wild-type enzyme in sucrose-containing solutions. The value of kcat for the sluggish mutant was unaffected by varying solvent viscosity. However, sucrose did have a slight activating effect on mutant enzyme efficiency. In the presence of the polymeric viscosogens poly(ethylene glycol) and Ficoll, no effect on kcat or kcat/Km for the wild-type enzyme was observed. These results are consistent with both substrate binding and product dissociation being partially rate-determining in both directions. The viscosity variation method was used to estimate the rate constants comprising the steady-state expressions for kcat and kcat/Km. The rate constant for the conversion of bound (R)-mandelate to bound (S)-mandelate (k2) was found to be 889 +/- 40 s(-1) compared with a value of 654 +/- 58 s(-1) for kcat in the same direction. From the temperature dependence of Km (shown to equal K(S)), k2, and the rate constant for the uncatalyzed reaction [Bearne, S. L., and Wolfenden, R. (1997) Biochemistry 36, 1646-1656], we estimated the enthalpic and entropic changes associated with substrate binding (DeltaH = -8.9 +/- 0.8 kcal/mol, TDeltaS = -4.8 +/- 0.8 kcal/mol), the activation barrier for conversion of bound substrate to bound product (DeltaH# = +15.4 +/- 0.4 kcal/mol, TDeltaS# = +2.0 +/- 0.1 kcal/mol), and transition state stabilization (DeltaH(tx) = -22.9 +/- 0.8 kcal/mol, TDeltaS(tx) = +1.8 +/- 0.8 kcal/mol) during mandelate racemase-catalyzed racemization of (R)-mandelate at 25 degrees C. Although the high proficiency of mandelate racemase is achieved principally by enthalpic reduction, there is also a favorable and significant entropic contribution.     

Binding of the recombinant proteinase inhibitor eglin c from leech Hirudo medicinalis to human leukocyte elastase, bovine alpha-chymotrypsin and subtilisin Carlsberg: thermodynamic study

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the recombinant proteinase inhibitor eglin c from leech Hirudo medicinalis to human leukocyte elastase (EC 3.4.21.37), bovine alpha-chymotrypsin (EC 3.4.21.1) and subtilisin Carlsberg (EC 3.4.21.14) has been investigated. On lowering the pH from 9.5 to 4.5, values of Ka for eglin c binding to the serine proteinases considered decrease thus reflecting the acid-pK shift of the invariant histidyl catalytic residue (His57 in human leukocyte elastase and bovine alpha-chymotrypsin, and His64 in subtilisin Carlsberg) from congruent to 6.9, in the free enzymes, to congruent to 5.1, in the enzyme:inhibitor adducts. At pH 8.0, values of the apparent thermodynamic parameters for eglin c binding are: human leukocyte elastase - Ka = 1.0 x 10(10) M-1, delta G phi = -13.4 kcal/mol, delta H phi = +1.8 kcal/mol, and delta S phi = +52 entropy units; bovine alpha-chymotrypsin -Ka = 5.0 x 10(9) M-1, delta G phi = -13.0 kcal/mol, delta H phi = +2.0 kcal/mol, and delta S phi = +51 entropy units; and subtilisin Carlsberg - Ka = 6.6 x 10(9) M-1, delta G phi = -13.1 kcal/mol, delta H phi = +2.0 kcal/mol, and delta S phi = +51 entropy units (values of Ka, delta G phi and delta S phi were obtained at 21 degrees C; values of delta H phi were temperature independent over the range explored, i.e. between 10 degrees C and 40 degrees C; 1 kcal = 4184J).(ABSTRACT TRUNCATED AT 250 WORDS)     

Large increases in general stability for subtilisin BPN' through incremental changes in the free energy of unfolding

Six individual amino acid substitutions at separate positions in the tertiary structure of subtilisin BPN' (EC 3.4.21.14) were found to increase the stability of this enzyme, as judged by differential scanning calorimetry and decreased rates of thermal inactivation. These stabilizing changes, N218S, G169A, Y217K, M50F, Q206C, and N76D, were discovered through the use of five different investigative approaches: (1) random mutagenesis; (2) design of buried hydrophobic side groups; (3) design of electrostatic interactions at Ca2+ binding sites; (4) sequence homology consensus; and (5) serendipity. Individually, the six amino acid substitutions increase the delta G of unfolding between 0.3 and 1.3 kcal/mol at 58.5 degrees C. The combination of these six individual stabilizing mutations together into one subtilisin BPN' molecule was found to result in approximately independent and additive increases in the delta G of unfolding to give a net increase of 3.8 kcal/mol (58.5 degrees C). Thermodynamic stability was also shown to be related to resistance to irreversible inactivation, which included elevated temperatures (65 degrees C) or extreme alkalinity (pH 12.0). Under these denaturing conditions, the rate of inactivation of the combination variant is approximately 300 times slower than that of the wild-type subtilisin BPN'. A comparison of the 1.8-A-resolution crystal structures of mutant and wild-type enzymes revealed only independent and localized structural changes around the site of the amino acid side group substitutions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Investigation of the functional contributions of invariant serine residues in yeast mevalonate diphosphate decarboxylase

Alignment of more than 20 deduced sequences for mevalonate diphosphate decarboxylase (MDD) indicates that serines 34, 36, 120,121, 153, and 155 are invariant residues that map within a proposed interdomain active site cleft. To test possible active site roles for these invariant serines, each has been mutated to alanine. S34A exhibits limited solubility and impaired binding of the fluorescent ATP analogue, trinitrophenyl-ATP (TNP-ATP), suggesting that Ser-34 substitution destabilizes proper enzyme folding. All other serine mutants retain structural integrity, as indicated by their ability to bind TNP-ATP at levels comparable to wild-type enzyme. S153A exhibits a 18-fold inflation in K(d) for Mg-ATP, as indicated by competitive displacement of TNP-ATP; the enzyme also is characterized by a 35-fold inflation in K(m) for Mg-ATP. S155A exhibits a 26-fold inflation in K(m) for Mg-ATP, but competitive displacement of TNP-ATP indicates only a 2-fold inflation in K(d) for this substrate. S155A exhibits both a 16-fold inflation in K(m) for mevalonate diphosphate and a 14-fold inflation in K(i(slope)) for the substrate analogue, diphosphoglycolylproline. These observations suggest roles for Ser-153 and Ser-155 in substrate binding. Catalytic consequences of mutating invariant serines 36, 120, 153, and 155 are modest (<8-fold diminution in k(cat)). In contrast, S121A, which exhibits only modest changes in K(d) for Mg-ATP and K(m) for mevalonate diphosphate, is characterized by a >42,000-fold diminution in k(cat), indicating the critical involvement of Ser-121 in reaction catalysis. The selective involvement of the latter of two tandem serine residues (Ser-120, Ser-121) in a conserved sequence motif suggests mechanistic similarities within the GHMP kinase superfamily of proteins.     

Incorporation of a stabilizing Ca(2+)-binding loop into subtilisin BPN'.

A rational approach was taken to improve the stability of subtilisin BPN' to autoproteolysis. Two sites of autoproteolysis were identified by isolation of early autolysis products and amino-terminal sequence analysis. These studies showed that subtilisin rapidly cleaves Ala48-Ser49 and Ser163-Thr164 peptide bonds at elevated temperatures. These two sites appear in regions of high mobility as estimated from crystallographic B-factors and are in extended surface loops. To improve the resistance to thermal-induced autolysis, we replaced sequences around these two sites with sequences derived from a thermophilic homologue of subtilisin, thermitase. Thermitase contains a Ca(2+)-binding site in the region surrounding Ser49. When the Ca(2+)-binding segment of thermitase corresponding to residues 45-63 of subtilisin BPN' was installed into subtilisin BPN', the chimeric protein gained the ability to bind another Ca2+ with moderate affinity (Kd approximately 100 microM). This enzyme had the same kcat as wild-type, had a KM value 8-fold larger than wild-type, and was slightly less stable to thermal inactivation in EDTA. However, in 10 mM CaCl2, the mutant subtilisin BPN' was 10-fold more stable to irreversible inactivation at 60 degrees C than wild-type subtilisin BPN' as measured by residual activity against the substrate sAAPF-pna. Next, mutations and deletions derived from thermitase were introduced near the second autolysis loop in subtilisin BPN' (residues 158-165). However, all of these mutants were less stable than wild-type subtilisin. Thus, some (but not all) mutations derived from a thermophilic homologue near sites of autolysis can be stabilizing to a mesophilic protease.     

The role of Pro-239 in the catalysis and heat stability of subtilisin E

Site-directed mutagenesis was employed to analyze the role of an alpha-helix containing catalytic Ser-221 of subtilisin E. Pro-239 located at the carboxy-terminal end of the alpha-helix was first replaced with Gly to examine the role of Pro-239 in the catalysis and stability of subtilisin E. The mutation was found to decrease both the catalytic rate (kcat) and the heat stability. This result strongly suggests that Pro-239 plays an important role in the maintenance of the alpha-helix, affecting the functioning of the active site. Various amino acid substitutions at position 239 were attempted to obtain the active subtilisins from Gly-239 subtilisin. Lys- and Arg-substitutions were found to result in more active and stable subtilisins than the Gly-239 subtilisin. In particular, the Arg-239 mutant showed enhanced heat stability compared with the wild type. These results demonstrate the important role of the alpha-helix containing catalytic Ser-221 in the catalysis as well as in the heat stability of subtilisin.     

Engineering subtilisin and its substrates for efficient ligation of peptide bonds in aqueous solution

Protein engineering techniques were used to construct a derivative of the serine protease subtilisin that ligates peptides efficiently in water. The subtilisin double mutant in which the catalytic Ser221 was converted to Cys (S221C) and Pro225 converted to Ala (P225A) has 10-fold higher peptide ligase activity and at least 100-fold lower amidase activity than the singly mutated thiolsubtilisin (S221C) that was previously shown to have some peptide ligase activity [Nakatsuka, T., Sasaki, T., & Kaiser, E.T. (1987) J. Am. Chem. Soc. 109, 3808-3810]. A 1.5-A X-ray crystal structure of an oxidized derivative of the double mutant (S221C/P225A) supports the protein design strategy in showing that the P225A mutation partly relieves the steric crowding expected from the S221C substitution, thus accounting for its improved catalytic efficiency. Stable and synthetically reasonable alkyl ester peptide substrates were prepared that rapidly acylate the S221C/P225A enzyme, and aminolysis of the resulting thioacyl-enzyme intermediate by various peptides is strongly preferred over hydrolysis. The efficiency of aminolysis is relatively insensitive to the sequence of the first two residues in the acyl acceptor peptide whose alpha-amino group attacks the thioacyl-enzyme. To obtain greater flexibility in the choice of coupling sites, a set of three additional peptide ligases were engineered by introducing mutations into the parent ligase (S221C/P225A) that were previously shown to change the specificity of subtilisin for the residue nearest the acyl bond (the P1 residue). The specificity properties of the parent ligase and derivatives of it paralleled those of wild type and corresponding specificity variants.(ABSTRACT TRUNCATED AT 250 WORDS)     

The roles of surface loop insertions and disulfide bond in the stabilization of thermophilic WF146 protease

Thermophilic WF146 protease possesses four surface loop insertions and a disulfide bond, resembling its psychrophilic (subtilisins S41 and S39) and mesophilic (subtilisins SSII and sphericase) homologs. Deletion of the insertion 3 (positions 193-197) or insertion 4 (positions 210-221) of WF146 protease resulted in a significant decrease of the enzyme stability. In addition, substitution of the residues Pro211 and Ala212 or residue Glu221 which localized in the vicinity of a Ca(2+) binding site of the enzyme by the corresponding residues in subtilisin S41 remarkably reduced the half-life of the enzyme at 70 degrees C, suggesting that the three residues contributed to the thermostability of the enzyme, probably by enhancing the affinity of enzyme to Ca(2+). In the presence of dithiothreitol, the WF146 protease suffered excessive autolysis, indicating that the Cys52-Cys65 disulfide bond played a critical role in stabilizing the WF146 protease against autolysis. The autolytic cleavage sites of the WF146 protease were identified to locate between residues Asn63-Gly64 and Cys65-Ala66 by N-terminal amino acid analysis of the autolytic product. It was noticed that the effect of the autolytic cleavage at Asn63-Gly64 could be compensated by the disulfide bond Cys52-Cys65 under non-reducing condition, and the disulfide bond cross-linked autolytic product remained active. The apparent stabilization effect of the disulfide bond Cys52-Cys65 in the WF146 protease might provide a rational basis for improving the stability of subtilase against autolysis by protein engineering.     

The structural bases of antibiotic resistance in the clinically derived mutant beta-lactamases TEM-30, TEM-32, and TEM-34

Widespread use of beta-lactam antibiotics has promoted the evolution of beta-lactamase mutant enzymes that can hydrolyze ever newer classes of these drugs. Among the most pernicious mutants are the inhibitor-resistant TEM beta-lactamases (IRTs), which elude mechanism-based inhibitors, such as clavulanate. Despite much research on these IRTs, little is known about the structural bases of their action. This has made it difficult to understand how many of the resistance substitutions act as they often occur far from Ser-130. Here, three IRT structures, TEM-30 (R244S), TEM-32 (M69I/M182T), and TEM-34 (M69V), are determined by x-ray crystallography at 2.00, 1.61, and 1.52 A, respectively. In TEM-30, the Arg-244 --> Ser substitution (7.8 A from Ser-130) displaces a conserved water molecule that usually interacts with the beta-lactam C3 carboxylate. In TEM-32, the substitution Met-69 --> Ile (10 A from Ser-130) appears to distort Ser-70, which in turn causes Ser-130 to adopt a new conformation, moving its O gamma further away, 2.3 A from where the inhibitor would bind. This substitution also destabilizes the enzyme by 1.3 kcal/mol. The Met-182 --> Thr substitution (20 A from Ser-130) has no effect on enzyme activity but rather restabilizes the enzyme by 2.9 kcal/mol. In TEM-34, the Met-69 --> Val substitution similarly leads to a conformational change in Ser-130, this time causing it to hydrogen bond with Lys-73 and Lys-234. This masks the lone pair electrons of Ser-130 O gamma, reducing its nucleophilicity for cross-linking. In these three structures, distant substitutions result in accommodations that converge on the same point of action, the local environment of Ser-130.     

Role of serine 214 and tyrosine 171, components of the S2 subsite of alpha-lytic protease, in catalysis.

The function of a hydrogen bond network, comprised of the hydroxyl groups of Tyr 171 and Ser 214, in the hydrophobic S2 subsite of alpha-lytic protease, was investigated by mutagenesis and the kinetics of a substrate analog series. To study the catalytic role of the Tyr 171 and Ser 214 hydroxyl groups, Tyr 171 was converted to phenylalanine (Y171F) and Ser 214 to alanine (S214A). The double mutant (Y171F: S214A) also was generated. The single S214A and double Y171F:S214A mutations cause differential effects on catalysis and proenzyme processing. For S214A, kcat/Km is (4.9 x 10(3))-fold lower than that of wild type and proenzyme processing is blocked. For the double mutant (Y171F:S214A), kcat/Km is 82-fold lower than that of wild type and proenzyme processing occurs. In Y171F, kcat/Km is 34-fold lower than that of wild type, and the proenzyme is processed. The data indicate that Ser 214, although conserved among serine proteases and hydrogen bonded to the catalytic triad [Brayer, G. D., Delbaere, L. T. J., & James, M. N. G. (1979) J. Mol. Biol. 131, 743], is not essential for catalytic function in alpha-lytic protease. A substrate series (in which peptide length is varied) established that the mutations (Y171F and Y171F:S214A) do not alter enzyme-substrate interactions in subsites other than S2. The pH dependence of kcat/Km for Y171F and Y171F:S214A has changed less than 0.5 unit from that of wild type; this suggests the catalytic triad is unperturbed. In wild type, hydrophobic interactions at S2 increase kcat/Km by up to (1.2 x 10(3))-fold with no effect on Km.(ABSTRACT TRUNCATED AT 250 WORDS)     

Substitutions in the active site of chloramphenicol acetyltransferase: role of a conserved aspartate

The role of conserved Asp-199 in chloramphenicol acetyltransferase (CAT) has been investigated by site-directed mutagenesis. Substitution of Asp-199 by alanine results in a thermolabile mutant enzyme (Ala-199 CAT) with reduced kcat(13-fold) but similar Km values to wild type CAT. Replacement by asparagine gives rise to a thermostable mutant enzyme (Asn-199 CAT) with much reduced kcat(1500-fold). Furthermore, Asn-199 CAT shows anomalous inactivation kinetics with the affinity reagent 3-(bromo-acetyl)chloramphenicol. These results favor a structural role for Asp-199 rather than a catalytic one, in keeping with crystallographic evidence for involvement of Asp-199 in a tight salt bridge with Arg-18. Replacement of Arg-18 by valine results in a mutant enzyme (Val-18 CAT) with similar properties to Ala-199 CAT. The catalytic imidazole of His-19 appears to be conformationally constrained by hydrogen bonding between N1-H and the carbonyl oxygen of the same residue and by ring stacking with Tyr-25.