Elementary steps in the reaction mechanism of chicken liver fatty acid synthase. pH dependence of NADPH binding and isotope rate effect for beta-ketoacyl reductase

The stopped flow method has been used to determine the pH dependence of the kinetics of the binding of NADPH to chicken liver fatty acid synthase over the pH range 6.0-8.5. The kinetics is consistent with a one-step binding mechanism, and the pH dependence of the second order rate constant indicates that an ionizable group either on the enzyme or on NADPH with a pK alpha of 6.1 is of importance in the binding process. The isotope rate effects have been determined for the steady state reaction with (S)- and (R)-[4-2H] NADPH as substrates and are very small. The pH dependence of the rate constant characterizing the reduction of acetoacetyl by NADPH on the enzyme (beta-ketoacyl reductase) and the isotope rate effects on this constant with (S)-[4-2H]NADPH as substrate also have been measured with the stopped flow method. A small pH-dependent isotope rate effect is found; these results suggest hydride transfer is not rate limiting for the beta-ketoacyl reductase reaction on the enzyme surface. The pH dependence of this rate constant is bell shaped and is very similar to that of the turnover number for the overall reaction; this suggests that the beta-ketoacyl reductase reaction may be partially rate limiting for the overall reaction when the enzyme is saturated with substrates.     

Dihydrofolate reductase: control of the mode of substrate binding by aspartate 26

The complex of Lactobacillus casei dihydrofolate reductase with the substrate folate and the coenzyme NADP+ has been shown to exist in solution as a mixture of three slowly interconverting conformations whose proportions are pH-dependent and which differ in the orientation of the pteridine ring of the substrate in the binding site. The Asp26----Asn mutant of L. casei dihydrofolate reductase has been prepared by oligonucleotide-directed mutagenesis and studied by one- and two-dimensional 1H-NMR spectroscopy. NMR studies of the mutant enzyme--folate--NADP+ complex show that this exists to greater than 90% in a single conformation over the pH* range 5-7.1. The single conformation observed corresponds to conformation I (the 'methotrexate-like' conformation) of the wild-type enzyme--folate--NADP+ complex. These observations demonstrate that Asp26 is the ionizable group controlling the pH-dependence of the conformational equilibrium seen in the wild-type enzyme.     

Role of aspartate 27 in the binding of methotrexate to dihydrofolate reductase from Escherichia coli

Dihydrofolate reductase from wild-type Escherichia coli (WT-ECDHFR) and from a mutant enzyme in which aspartate 27 is replaced by asparagine have been compared with respect to the binding of the inhibitor methotrexate (MTX). Although the Asp27----Asn substitution causes only small changes in the association rate constants (kon) for the formation of binary and ternary (with NADPH) complexes, the dissociation rate constants for these complexes (koff) are increased for the mutant enzyme by factors of about 5- and 100-fold, respectively, at pH 7.65. In binding experiments, the initial MTX binary and ternary complexes of the mutant enzyme were found to undergo relatively rapid isomerization (kobs approximately 17 and 145 s-1, respectively). Although such rapid isomerization of complexes of WT-ECDHFR could not be detected in binding experiments, evidence of a slow isomerization (k = 4 x 10(-3) s-1) of the ternary WT-ECDHFR.MTX.NADPH complex was obtained from progress of inhibition experiments. This slow isomerization increases binding of MTX to WT-ECDHFR only 2.4-fold (much less than previously estimated). From presently available data, we could not determine the contribution of the rapid isomerization of complexes to the binding of MTX to the mutant enzyme. The Asp27----Asn substitution increases the overall dissociation constant (KD) 9-fold for the binary complex and 85-fold for the ternary complex. When it is also taken into account that a proton ultimately derived from the solvent must be added to MTX bound to the WT enzyme, but not to MTX bound to the mutant enzyme, these increases in KD for the mutant enzyme correspond to decreases in binding energy for MTX of 3.9 and 5.2 kcal/mol at pH 7.65 for the binary and ternary complexes, respectively.     

Effects of conversion of an invariant tryptophan residue to phenylalanine on the function of human dihydrofolate reductase

The binding site residue Trp-24 is conserved in all vertebrate and bacterial dihydrofolate reductases of known sequence. To determine its effects on enzyme properties, a Trp-24 to Phe-24 mutant (W-24-F) of human dihydrofolate reductase has been constructed by oligodeoxynucleotide site-directed mutagenesis. The W-24-F mutant enzyme appears to have a more open or flexible conformation as compared to the wild-type human dihydrofolate reductase on the basis of results of a number of studies. These studies include competitive ELISA using peptide-specific antibodies against human dihydrofolate reductase, thermal stability, and protease susceptibility studies of both mutant W-24-F and wild-type enzymes. It is concluded that Trp-24 is important for maintaining the structural integrity of the native enzymes. Changes in relative fluorescence quantum yield indicate that Trp-24 is buried and its fluorescence quenched relative to the other two tryptophan residues in the wild-type human reductase. Kinetic studies indicate that kcat values for W-24-F are increased in the pH range of 4.5-8.5 with a 5-fold increase at pH 7.5 as compared to the wild-type enzyme. However, the catalytic efficiency of W-24-F decreases rapidly as the pH is increased from 7.5 to 9.5. The Km values for dihydrofolate are also increased for W-24-F in the pH range of 4.5-9.5 with a 30-fold increase at pH 7.5, while the Km value for NADPH increases only ca. 1.4-fold at pH 7.5 as compared to the wild type.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of the reaction catalyzed by dihydrofolate reductase from Escherichia coli: pH and deuterium isotope effects with NADPH as the variable substrate

The variations with pH of the kinetic parameters and primary deuterium isotope effects for the reaction of NADPH with dihydrofolate reductase from Escherichia coli have been determined. The aims of the investigations were to elucidate the chemical mechanism of the reaction and to obtain information about the location of the rate-limiting steps. The V and V/KNADPH profiles indicate that a single ionizing group at the active center of the enzyme must be protonated for catalysis, whereas the Ki profiles show that the binding of NADPH to the free enzyme and of ATP-ribose to the enzyme-dihydrofolate complex is pH independent. From the results of deuterium isotope effects on V/KNADPH, it is concluded that NADPH behaves as a sticky substrate. It is this stickiness that raises artificially the intrinsic pK value of 6.4 for the Asp-27 residue of the enzyme-dihydrofolate complex [Howell, E. E., Villafranca, J. E., Warren, M. S., Oatley, S. J., & Kraut, J. (1986) Science (Washington, D.C.) 231, 1123] to an observed value of 8.9. Thus, the binary enzyme complex is largely protonated at neutral pH. The elevation of the intrinsic pK value of 6.4 for the ternary enzyme-NADPH-dihydrofolate complex to 8.5 is not due to the kinetic effects of substrates. Rather, it is the consequence of the lower, pH-independent rate of product release and the faster pH-dependent catalytic step. At neutral pH, the proportion of enzyme present as a protonated ternary enzyme-substrate complex is sufficient to keep catalysis faster than product release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reversible dissociation of dimeric tyrosyl-tRNA synthetase by mutagenesis at the subunit interface

Dimeric tyrosyl-tRNA synthetase from Bacillus stearothermophilus exhibits half-of-the-sites reactivity and negative cooperativity in binding of tyrosine. Protein engineering has been applied to the enzyme to determine whether it can be reversibly dissociated into monomers and if the monomers are active. The target for mutation is the residue Phe-164. The side chain of Phe-164 in one subunit interacts with its symmetry-related partner in the other. Mutation of Phe-164----Asp-164 gives a mutant [TyrTS(Asp-164)] that undergoes dissociation at high pH when the aspartate residues are ionized. The monomer is inactive and does not bind tyrosine. Dissociation is enhanced at low concentrations of enzyme by a mass action effect. Kinetic and binding measurements on TyrTS(Asp-164) with tyrosine and tyrosyl adenylate show that the monomer has very weak affinity for these ligands. Accordingly, dimerization is favored by high concentrations of tyrosine and ATP since the dimeric form has a high affinity for the ligands. The presence of tRNA does not encourage dimer formation, and so it must bind to the monomer. TyrTS(Asp-164) is fully active at pH 6 where dimerization is favored but has low activity at pH 7.8 where dissociation is favored. It should now prove possible to engineer heterodimers that may be used to investigate the subunit interactions further.     

Role of aspartate 27 of dihydrofolate reductase from Escherichia coli in interconversion of active and inactive enzyme conformers and binding of NADPH

The apoenzyme of wild-type (WT) dihydrofolate reductase (DHRF) from Escherichia coli exists in two conformational states, Et and Ew, which differ in affinity for NADPH and in kinetic competence. Dissociation constants for the binary complex of NADPH with the two conformers differ by over 100-fold (KDt = 0.17 microM, KDw = 22 microM). Rate constants governing the interconversion of conformers are small (t1/2 for Ew----Et = 71 s), and since Ew is not catalytically competent, this conversion is accompanied by an increase in catalytic velocity. The equilibrium proportion of Et in the absence of ligands is 63%, but binding of NADPH greatly increases this proportion, and t1/2 for conversion of Ew.NADPH to Et.NADPH is 30 s. This conformational equilibrium has also been examined in mutant enzyme in which aspartate 27 is replaced by asparagine (D27N E. coli DHFR). Although ASp27 is an active site residue, it does not interact directly with bound NADPH, and in the mutant the rate constant for NADPH binding to Et is unchanged as are the dissociation constants for NADPH complexes with Et or Ew. However, for mutant apoenzyme, the proportion of Et is decreased to 18% in the absence of ligands so that the overall KD for NADPH is increased (0.15 microM for WT E. coli DHFR, 0.68 microM for D27N E. coli DHFR). The lower proportion of Et is due to a decreased rate for Ew----Et (t1/2 = 221 s) and an increased rate for Et----Ew (t1/2 = 50 s versus 120 s for WT E. coli DHFR).     

Studies on the interaction of NADPH with Rhodobacter sphaeroides biotin sulfoxide reductase

Rhodobacter sphaeroides biotin sulfoxide reductase (BSOR) contains the bis(molybdopterin guanine dinucleotide)molybdenum cofactor and catalyzes the reduction of D-biotin-D-sulfoxide to biotin. This protein is the only member of the dimethyl sulfoxide reductase family of molybdopterin enzymes that utilizes NADPH as the direct electron donor to the catalytic Mo center. Kinetic studies using stopped-flow spectrophotometry indicate that BSOR reduction by NADPH (>1000 s(-1)) is faster than steady-state turnover (440 s(-1)) and has shown that BSOR reduction occurs in concert with NADPH oxidation with no indication of a Mo(V) intermediate species. Because no crystallographic structure is currently available for BSOR, a protein structure was modeled using the structures for R. sphaeroides dimethyl sulfoxide reductase, Rhodobacter capsulatus dimethyl sulfoxide reductase, and Shewanella massilia trimethylamine N-oxide reductase as the templates. A potential NADPH-binding site was identified and tested by site-directed mutagenesis of residues within the area. Mutation of Arg137 or Asp136 reduced the ability of NADPH to serve as the electron donor to BSOR, indicating that the NADPH-binding site in BSOR is located in the active-site funnel of the putative structure where it can directly reduce the Mo center. Along with kinetic and spectroscopic data, the location of this binding site supports a direct hydride transfer mechanism for NADPH reduction of BSOR.     

Studies on the reactivity of the essential sulfhydryl groups as a conformational probe for the fatty acid synthetase of chicken liver. Inactivation by 5,5'-dithiobis-(2-nitrobenzoic acid) and intersubunit cross-linking of the inactivated enzyme

Fatty acid synthetase of chicken liver is rapidly and reversibly inactivated by 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) at a rate (k2 = 132 mM-1 S-1 in 3 mM EDTA, 1% (v/v) glycerol, pH 7.0, at 25 degrees C) up to 2200 times higher than the reaction of this reagent with simple thiol compounds. The inactivation is caused by the reaction of the phosphopantetheine SH group, since it is protected competitively by either acetyl- or malonyl-CoA, and since the inactivated enzyme is unreactive with the phosphopantetheine label chloroacetyl-CoA but reactive with the cysteine reagent 1,3-dibromopropanone. Moreover, chloroacetyl-CoA prevents the modification of the rapidly reacting essential SH group by DTNB. The number of SH groups involved in inactivation was determined by correlating activity loss with the extent of reaction and by stopped-flow analysis of substrate (or chloroacetyl-CoA) protection. Values between 0.91 and 1.15 SH groups/dimer were obtained, indicating the presence of substoichiometric amounts of the prosthetic group in the fatty acid synthetase preparations used in this study. Inactivation of the synthetase by DTNB is strongly inhibited by increasing salt concentration and protected noncompetitively by NADP+ and NADPH. Treatment of the enzyme inactivated at low salt by salt, NADP+, or NADPH also effectively reduced cross-linking between enzyme subunits. The parallel effects of these treatments on the reaction with DTNB and subsequent dimerization are consistent with a minimum model of two discreet conformation states for fatty acid synthetase. In the low salt conformer, the phosphopantetheine and cysteine SH groups are juxtaposed, and the DTNB reaction (k2 approximately 132 mM-1 S-1) and dimerization are both facilitated. Transition to the high salt conformer by the above treatments is accompanied by an approximately 20-fold reduction of reactivity with DTNB (k2 = 6.8 mM-1 S-1) and reduced dimerization, due to spatial separation of the SH groups. During palmitate synthesis, the enzyme may oscillate between these conformation states to permit the reaction of intermediates at different active sites. Results obtained by studies on the effect of pH on DTNB inactivation implicate a pK of 5.9-6.1 for the essential SH group independent of salt concentration. This value is 1.5-1.8 pH units lower than the pK of 7.6-7.7 for CoA and may explain the 23-fold increase of the rate constant from a value of 0.3 mM-1 S-1 for CoA to that of the high salt conformer.     

A second-site mutation at phenylalanine-137 that increases catalytic efficiency in the mutant aspartate-27----serine Escherichia coli dihydrofolate reductase

The adaptability of Escherichia coli dihydrofolate reductase (DHFR) is being explored by identifying second-site mutations that can partially suppress the deleterious effect associated with removal of the active-site proton donor aspartic acid-27. The Asp27----serine mutant DHFR (D27S) was previously characterized and the catalytic activity found to be greatly decreased at pH 7.0 [Howell et al. (1986) Science 231, 1123-1128]. Using resistance to trimethoprim (a DHFR inhibitor) in a genetic selection procedure, we have isolated a double-mutant DHFR gene containing Asp27----Ser and Phe137----Ser mutations (D27S+F137S). The presence of the F137S mutation increases kcat approximately 3-fold and decreases Km(DHF) approximately 2-fold over D27S DHFR values. The overall effect on kcat/Km(DHF) is a 7-fold increase. The D27S+F137S double-mutant DHFR is still 500-fold less active than wild-type DHFR at pH 7. Surprisingly, Phe137 is approximately 15 A from residue 27 in the active site and is part of a beta-bulge. We propose the F137S mutation likely causes its catalytic effect by slightly altering the conformation of D27S DHFR. This supposition is supported by the observation that the F137S mutation does not have the same kinetic effect when introduced into the wild-type and D27S DHFRs, by the altered distribution of two conformers of free enzyme [see Dunn et al. (1990)] and by a preliminary difference Fourier map comparing the D27S and D27S+F137S DHFR crystal structures.     

High pressure NMR reveals active-site hinge motion of folate-bound Escherichia coli dihydrofolate reductase

A high-pressure (15)N/(1)H two-dimensional NMR study has been carried out on folate-bound dihydrofolate reductase (DHFR) from Escherichia coli in the pressure range between 30 and 2000 bar. Several cross-peaks in the (15)N/(1)H HSQC spectrum are split into two with increasing pressure, showing the presence of a second conformer in equilibrium with the first. Thermodynamic analysis of the pressure and temperature dependencies indicates that the second conformer is characterized by a smaller partial molar volume (DeltaV = -25 mL/mol at 15 degrees C) and smaller enthalpy and entropy values, suggesting that the second conformer is more open and hydrated than the first. The splittings of the cross-peaks (by approximately 1 ppm on (15)N axis at 2000 bar) arise from the hinges of the M20 loop, the C-helix, and the F-helix, all of which constitute the major binding site for the cofactor NADPH, suggesting that major differences in conformation occur in the orientations of the NADPH binding units. The Gibbs free energy of the second, open conformer is 5.2 kJ/mol above that of the first at 1 bar, giving an equilibrium population of about 10%. The second, open conformer is considered to be crucial for NADPH binding, and the NMR line width indicates that the upper limit for the rate of opening is 20 s(-)(1) at 2000 bar. These experiments show that high pressure NMR is a generally useful tool for detecting and analyzing "open" structures of a protein that may be directly involved in function.     

Atypical transient state kinetics of recombinant human dihydrofolate reductase produced by hysteretic behavior. Comparison with dihydrofolate reductases from other sources

The transient state kinetics of catalysis for dihydrofolate reductase (DHFR) from several enzyme sources including highly purified recombinant human enzyme (rHDHFR) have been examined. Like DHFR from Escherichia coli, the enzyme from Lactobacillus casei, and isoenzyme 2 from Streptococcus faecium exhibit a slow increase in activity upon addition of substrates to enzyme. No slow hysteresis of this type was detected with recombinant human DHFR (rHDHFR) or DHFR from chicken or bovine liver or L1210 mouse leukemia cells (MDHFR). In contrast, both rHDHFR and MDHFR exhibited a very rapid decrease in activity (t1/2 = 30 and 20 ms, respectively) during a phase that occurred after the first turnover of the enzyme but before establishment of the steady state. This intermediate phase was not observed for the bacterial enzymes or the avian enzyme, nor was it observed with a mutant of rHDHFR in which Phe-31 has been replaced by leucine. For rHDHFR the intermediate phase is not a consequence of product inhibition, substrate depletion, or enzyme instability. It may therefore be concluded that this unusual transient state kinetic behavior results from the existence of two conformers of the enzyme, one of which has a higher turnover number than the other with the equilibrium shifting in favor of the less active conformer during the course of catalysis. The equilibrium is particularly favorable for the less active conformer when NADP is present in the active site of rHDHFR, whereas bound tetrahydrofolate favors the more active conformer. The more active conformer has a 6-fold higher Km for dihydrofolate than does the less active conformer. The existence of these conformers is likely to produce cooperative behavior by rHDHFR in vivo.     

Human liver aldehyde reductase: pH dependence of steady-state kinetic parameters.

The pH dependence of steady-state parameters for aldehyde reduction and alcohol oxidation were determined in the human liver aldehyde reductase reaction. The maximum velocity of aldehyde reduction with NADPH or 3-acetyl pyridine adenine dinucleotide phosphate (3-APADPH) was pH independent at low pH but decreased at high pH with a pK of 8.9-9.6. The V/K for both nucleotides decreased below a pK of 5.7-6.2, as did the pKi of competitive inhibitors NADP and ATP-ribose, suggesting that the 2'-phosphate of the nucleotide has to be deprotonated for binding to the enzyme. The pK of the 2'-phosphate of NADPH appears to be perturbed in the ternary complexes to 5.2-5.4. The V/K for NADPH, the V/K for 3-APADPH, and the pKi of ATP-ribose also decreased above a pK of 9-10, suggesting interaction of the 2'-phosphate of the nucleotide with a protonated base, perhaps lysine. Since protonation of a residue with a pK of 8 (evident in V/K for DL-glyceraldehyde and V/K for L-gulonate versus pH profiles) appears to be essential for aldehyde reduction, and deprotonation for alcohol oxidation, this residue appears to act as a general acid-base catalyst. An additional anion binding site with a pK of 9.94 facilitates the binding of carboxylic substrates such as D-glucuronate. With NADPH as the coenzyme the primary deuterium isotope effects on V and V/K for NADPH were close to unity and pH independent, suggesting that the hydride transfer step is not rate determining over the experimental pH range. With 3-APADPH as the coenzyme, the maximum velocity, relative to NADPH was three- to four-fold lower. Isotope effects on V, V/K for 3-APADPH, and V/K for D-glucuronate were pH independent and equal to 2.2-2.8, indicating that the chemical step of the reaction is relatively insensitive to pH. These data suggest that substrates bind to both the protonated and the deprotonated forms of the enzyme, though only the protonated enzyme catalyzes aldehyde reduction and the deprotonated enzyme catalyzes alcohol oxidation. On the basis of these results a scheme for the chemical mechanism of aldehyde reductase is postulated.     

Probing the role of two hydrophobic active site residues in the human dihydrofolate reductase by site-directed mutagenesis

In the x-ray structure of the human dihydrofolate reductase, phenylalanine 31 and phenylalanine 34 have been shown to be involved in hydrophobic interactions with bound substrates and inhibitors. Using oligonucleotide-directed mutagenesis and a bacterial expression system producing the wild-type and mutant human dihydrofolate reductases at levels of 10% of the bacterial protein, we have constructed, expressed, and purified a serine 31 (S31) mutant and a serine 34 (S34) mutant. Fluorescence titration experiments indicated that S31 bound the substrate H2folate 10-fold tighter and the coenzyme NADPH 2-fold tighter than the wild-type human dihydrofolate reductase. The serine 31 mutation had little effect on the steady-state kinetic properties of the enzyme but produced a 100-fold increase in the dissociation constant (Kd) for the inhibitor methotrexate. The serine 34 mutant had much greater alterations in its properties than S31; specifically, S34 had a 3-fold reduction in the Km for NADPH, a 24-fold increase in the Km for H2folate, a 3-fold reduction in the overall reaction rate kcat, and an 80,000-fold increase in the Kd for methotrexate. In addition, the pH dependence of the steady-state kinetic parameters of S34 were different from that of the wild-type enzyme. These results suggest that phenylalanine 31 and phenylalanine 34 make very different contributions to ligand binding and catalysis in the human dihydrofolate reductase.     

An examination of the role of asp-177 in the His-Asp catalytic dyad of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase: X-ray structure and pH dependence of kinetic parameters of the D177N mutant enzyme

The role of Asp-177 in the His-Asp catalytic dyad of glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides has been investigated by a structural and functional characterization of the D177N mutant enzyme. Its three-dimensional structure has been determined by X-ray cryocrystallography in the presence of NAD(+) and in the presence of glucose 6-phosphate plus NADPH. The structure of a glucose 6-phosphate complex of a mutant (Q365C) with normal enzyme activity has also been determined and substrate binding compared. To understand the effect of Asp-177 on the ionization properties of the catalytic base His-240, the pH dependence of kinetic parameters has been determined for the D177N mutant and compared to that of the wild-type enzyme. The structures give details of glucose 6-phosphate binding and show that replacement of the Asp-177 of the catalytic dyad with asparagine does not affect the overall structure of glucose 6-phosphate dehydrogenase. Additionally, the evidence suggests that the productive tautomer of His-240 in the D177N mutant enzyme is stabilized by a hydrogen bond with Asn-177; hence, the mutation does not affect tautomer stabilization. We conclude, therefore, that the absence of a negatively charged aspartate at 177 accounts for the decrease in catalytic activity at pH 7.8. Structural analysis suggests that the pH dependence of the kinetic parameters of D177N glucose 6-phosphate dehydrogenase results from an ionized water molecule replacing the missing negative charge of the mutated Asp-177 at high pH. Glucose 6-phosphate binding orders and orients His-178 in the D177N-glucose 6-phosphate-NADPH ternary complex and appears to be necessary to form this water-binding site.     

One- and two-electron reduction of quinones by glutathione reductase

Yeast glutathione reductase (E.C. 1.6.4.2) catalyzes the oxidation of NADPH by p-quinones and ferricyanide with a maximal turnover number (TNmax) of 4-5 s-1.NADP+ stimulates the reaction and the TNmax/Km value of acceptors is reached at NADP+/NADPH greater than or equal to 100. TNmax is increased up to 30-33 s-1. The stimulatory effect of NADP+ may be associated with its complexation with the NADPH-binding site in the reduced enzyme (Kd = 40-60 microM). It is suggested that NADP+ shifts the electron density towards FAD in the two-electron-reduced enzyme and, evidently, changes its one-electron-reduction potentials, while quinones oxidize an equilibrium form of glutathione reductase containing reduced FAD. In the absence of NADP+ the reduction of quinones by glutathione reductase proceeds mainly in a two-electron manner. At NADP+/NADPH = 100 a one-electron reduction makes up 44% of the total process. At pH 6.0-7.0 the reduced forms of naphthoquinones undergo cyclic redox conversions. A hyperbolic dependence exists of the log TN/Km of quinones on their one-electron-reduction potentials.     

Hydrophobic amino acid residues in the acceptor binding site are main determinants for reaction mechanism and specificity of cyclodextrin-glycosyltransferase

Cyclodextrin-glycosyltransferases (CGTases) (EC ) preferably catalyze transglycosylation reactions with glucosyl residues as acceptor, whereas the homologous alpha-amylases catalyze hydrolysis reactions using water as acceptor. This difference in reaction specificity is most likely caused by the acceptor binding site. To investigate this in detail we altered the acceptor site residues Lys-232, Phe-183, Phe-259, and Glu-264 of Bacillus circulans strain 251 CGTase using site-directed mutagenesis. Lys-232 is of general importance for catalysis, which appears to result mainly from stabilization of the conformation of the loop containing the catalytic nucleophile Asp-229 and His-233, a residue that has been implied in transition state stabilization. Glu-264 contributes to the disproportionation reaction only, where it is involved in initial binding of the (maltose) acceptor. Phe-183 and Phe-259 play important and distinct roles in the transglycosylation reactions catalyzed by CGTase. Mutation of Phe-183 affects especially the cyclization and coupling reactions, whereas Phe-259 is most important for the cyclization and disproportionation reactions. Moreover, the hydrophobisity of Phe-183 and Phe-259 limits the hydrolyzing activity of the enzyme. Hydrolysis can be enhanced by making these residues more polar, which concomitantly results in a lower transglycosylation activity. A double mutant was constructed that yielded an enzyme preferring hydrolysis over cyclization (15:1), whereas the wild type favors cyclization over hydrolysis (90:1).     

Stabilization of a novel enzyme.substrate intermediate in the Y206F mutant of Candida albicans EBP1: evidence for acid catalysis

EBP1-catalyzed reduction of alpha,beta-unsaturated ketones and aldehydes is proposed to proceed via transfer of hydride from the flavin to the beta-position of the olefinic bond, concomitant with or followed by uptake of a proton at the alpha-position. Structural analysis suggests that this proton is donated from Tyr206, and, hence, a protein was constructed in which it was replaced by phenylalanine. The mutation results in a slightly less stable protein than the wild type that nevertheless retains the fundamental flavin and phenol binding properties of EBP1 characterized previously. The pH profile for binding of phenol was characterized over the pH range 6.5-9.5 and was found to be simpler than that for the wild-type enzyme. Most importantly, a pK(a) of 8.7 that is perturbed to 9.4 upon binding of phenol to the wild-type enzyme is missing in the mutant, allowing assignment of this pK(a) to the Y206 hydroxyl group. Additionally, the pK(a) of phenol is further lowered from its value of 10.0 in solution to approximately 6.4 in the active site of the mutant, as compared to 7.1 in the wild type. Together, these perturbations lead to an increase of approximately 35-fold in the binding affinity of the mutant for phenol at high pH relative to the affinity of the wild-type enzyme. As expected, the mutation has little effect on the reductive half-reaction, in which a hydride equivalent is transferred from NADPH to the flavin. In contrast, the reduction of trans-2-hexenal by the reduced enzyme is significantly affected. The results indicate formation of a previously unobserved charge-transfer (CT) complex following formation of the Michaelis complex between substrate and reduced enzyme and preceding reduction of the substrate, which occurs at a greatly reduced rate (>/=440-fold) relative to wild type. Thus, while the oxidative half-reaction with wild-type enzyme is limited by the rate of formation of the CT complex, it is the chemical step that is rate-limiting in the reaction with EBP1:Y206F, consistent with the role of this residue as a general acid.     

pH dependence of the binding constants of N-acetylglucosamine monomers to hen and turkey egg-white lysozymes.

The binding constants of N-acetylglucosamine (G1cNAc) and its methyl alpha- and beta- glycosides to hen and turkey egg-white lysozymes [EC 3.2.1.17], in the latter of which Asp 101 is replaced by Gly, were determined at various pH values by measuring changes in the circular dichroic (DC) band at 295 nm. The binding of beta-methyl-G1cNAc to turkey and hen lysozymes perturbed the pK value of Glu 35 from 6.0 to 6.5, the pK value of Asp 52 from 3.5 to 3.9, and the pK value of Asp 66 from 1.3 to 0.7. In addition, perturbation of the pK value of Asp 101 from 4.4 to 4.0 was observed in the binding of this saccharide to hen lysozyme. The binding of alpha-methyl-GlcNAc to hen and turkey lysozymes perturbed the pK value of Glu 35 to the alkaline side by about 0.5 pH unit, the pK value of Asp 66 to the acidic side by about 0.5 pH unit, and the pK value (4.4) of an ionizable group to the acidic side by about 0.6 pH unit. The last ionizable group was tentatively assigned to Asp 48. The pK value of Asp 52 was not perturbed by the binding of this saccharide. The pH dependence curves for the binding of GlcNAc to hen and turkey lysozymes were very similar and it was suggested that Asp 48, in addition to Asp 66, Asp 52, and Glu 35, is perturbed by the binding of GlcNAc.     

Construction of an altered proton donation mechanism in Escherichia coli dihydrofolate reductase

We have explored the substrate protonation mechanism of Escherichia coli dihydrofolate reductase by changing the location of the proton donor. A double mutant was constructed in which the proton donor of the wild-type enzyme, aspartic acid-27, has been changed to serine and simultaneously an alternative proton donor, glutamic acid, has replaced threonine at position 113. The active site of the resulting variant enzyme molecule should therefore somewhat resemble that proposed for the R67 plasmid-encoded dihydrofolate reductase [Matthews, D. A., Smith, S. L., Baccanari, D. P., Burchall, J. J., Oatley, S. J., & Kraut, J. (1986) Biochemistry 25, 4194]. At pH 7, the double-mutant enzyme has a 3-fold greater kcat and an unchanged Km(dihydrofolate) as compared with the single-mutant Asp-27----Ser enzyme described previously [Howell, E. E., Villafranca, J. E., Warren, M. S., Oatley, S. J., & Kraut, J. (1986) Science (Washington, D.C.) 231, 1123]. Additionally, its activity vs pH profiles together with observed deuterium isotope effects, suggest that catalysis depends on an acidic group with a pKa of 8. It is concluded that the dihydropteridine ring of a bound substrate molecule can indeed be protonated by a glutamic acid side chain at position 113 (instead of an aspartic acid side chain at position 27), but with greatly decreased efficiency: at pH 7, the double mutant still has a 25-fold lower kcat (1.2 s-1) and a 2900-fold lower kcat/km(dihydrofolate) (8.6 X 10(3) s-1 M-1) than the wild-type enzyme.