Site-directed mutagenesis in analysis of protein-protein interactions.
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Amino Acid Sequence↗
Biomedical subjects
J F Kirsch
Publications and source records attributed to J F Kirsch.
Reconstruction and testing of ancestral proteins.
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Animals↗
Contribution to catalysis and stability of the five cysteines in Escherichia coli aspartate aminotransferase. Preparation and properties of a cysteine-free enzyme.
The five cysteines, at positions 82, 191, 192, 270, and 401, of Escherichia coli aspartate aminotransferase (AATase) were, individually and in some combinations, converted to alanine by site-directed mutagenesis (C82A, C191A, C192A, C270A, C401A). Cys-191, which is conserved in all AATase isozymes, was mutated to serine as well (C191S). A quintuple mutant, with all cysteines converted to alanines (Quint), was also constructed. The effects of these single and multiple mutations were examined by steady-state kinetics and urea denaturation. The thermal stabilities of Quint and of the wild-type enzyme (WT) were determined by differential scanning calorimetry. The mutants had kcat values up to 50% greater than that of WT and KMAsp and KM alpha-KG values up to 1.5- and 3.3-fold higher than that of WT. The mutants C82A and C191A exhibit nearly the same CM in urea denaturation experiments as WT, while the other single mutants and Quint are less stable, with CM differences of up to 0.7 M urea. Quint is also less thermostable than WT, with a delta TM of 3.3-4.4 degrees C. Thus the five cysteine replacements yield small, but significant, changes in catalytic and denaturation parameters, but none of the cysteines was found to be essential. The changes manifested in the mutation of the conserved Cys-191 to alanine are no greater than those observed with the four nonconserved cysteines. We consider the evolutionary implications of these findings.
Amino Acid Sequence↗
Brønsted analysis of aspartate aminotransferase via exogenous catalysis of reactions of an inactive mutant.
Primary amines functionally replace lysine 258 by catalyzing both the 1,3-prototropic shift and external aldimine hydrolysis reactions with the inactive aspartate aminotransferase mutant K258A. This finding allows classical Brønsted analyses of proton transfer reactions to be applied to enzyme-catalyzed reactions. An earlier study of the reaction of K258A with cysteine sulfinate (Toney, M.D. & Kirsch, J.F., 1989, Science 243, 1485) provided a beta value of 0.4 for the 1,3-prototropic shift. The beta value reported here for the transamination of oxalacetate to aspartate is 0.6. The catalytic efficacy of primary amines is largely determined by basicity and molecular volume. The dependence of the rate constants for the reactions of K258A and K258M on amine molecular volume is nearly identical. This observation argues that the alkyl groups of the added amines do not occupy the position of the lysine 258 side chain in the wild type enzyme. Large primary C alpha and insignificant solvent deuterium kinetic isotope effects with amino acid substrates demonstrate that the amine nitrogen of the exogenous catalysts directly abstracts the labile proton in the rate-determining step.
Acetates↗
A rapid method for determination of endoproteinase substrate specificity: specificity of the 3C proteinase from hepatitis A virus.
The preferred amino acid residues at the P'1 and P'2 positions of peptide substrates of the 3C proteinase from hepatitis A virus (HAV-3C) have been determined by a rapid screening method. The enzyme was presented with two separate mixtures of N-terminal acetylated peptides, which were identical in sequence except for the amino acids at the P'1 or P'2 positions, where a set of 15 or 16 amino acids was introduced. Enzyme-catalyzed hydrolysis of the peptide mixtures generated free amino termini, which allowed direct sequence analysis by Edman degradation. The relative yield of each amino acid product in the appropriate sequencing cycle gave the amount of each substrate mixture component hydrolyzed. This allowed the simultaneous evaluation of the relative kcat/Km values for each component in the mixture. The peptide substrates preferred by the HAV-3C proteinase in the P'1 mixture were glycine, alanine, and serine. The enzyme has little specificity at P'2; only arginine and proline peptides were excluded as substrates. This method provides a rapid determination of the preferred residues for a peptide substrate and should be applicable to other endoproteinases.
3C Viral Proteases↗
The K258R mutant of aspartate aminotransferase stabilizes the quinonoid intermediate.
Lys-258 of aspartate aminotransferase forms a Schiff base with pyridoxal phosphate and is responsible for catalysis of the 1,3-prototropic shift central to the transamination reaction sequence. Substitution of arginine for Lys-258 stabilizes the otherwise elusive quinonoid intermediate, as assessed by the long wavelength absorption bands observed in the reactions of this mutant with several amino acid substrates. The external aldimine intermediate is not detectable during reactions of this mutant with amino acids, although the inhibitor alpha-methylaspartate does slowly and stably form this species. These results suggest that external aldimine formation is one of the rate-determining steps of the reaction. The pyridoxamine-5'-phosphate-like enzyme form (330-nm absorption maximum) is unreactive toward keto acid substrates, and the coenzyme bound to this species is not dissociable from the protein.
Amino Acid Sequence↗
Reengineering the catalytic lysine of aspartate aminotransferase by chemical elaboration of a genetically introduced cysteine.
The active-site essential catalytic residue of aspartate aminotransferase, Lys 258, has been converted to Cys (K258C) by site-directed mutagenesis. This mutant retains less than 10(-6) of the wild-type activity with L-aspartate. The deleted general base was functionally replaced by selective (with respect to the other five cysteines in wild type) aminoethylation of the introduced Cys 258 with (2-bromoethyl)amine following reversible protection of the nontarget sulfhydryl groups at different stages of unfolding. The chemically elaborated mutant (K258C-EA) is 10(5) times more reactive than is K258C and has a kcat value of approximately 7% of that of wild type (WT). Km and KI values are similar to those for WT. The acidic pKa controlling V/KAsp is shifted from 7.3 (WT) to 6.0 (mutant). V/K values for amino acids are approximately 3% of those found for WT, whereas they are approximately 20% for keto acids. The value of DV increases from 1.6 for WT to 3.4 for the mutant, indicating that C alpha proton abstraction constitutes a more significant kinetic barrier for the latter enzyme. A smaller, but still significant, increase in D(V/KAsp) from 1.9 in WT to 3.0 in the mutant shows that the forward and reverse commitment factors are inverted by the mutation. The acidic limb of the V/KAsp versus pH profile, is lowered by 1.3 pH units, probably reflecting the similar difference in the basicity of the epsilon-NH2 group in gamma-thialysine versus that in lysine.(ABSTRACT TRUNCATED AT 250 WORDS)
Alkylation↗
Tyrosine 70 fine-tunes the catalytic efficiency of aspartate aminotransferase.
The aspartate aminotransferase mutant Y70F exhibits kcat = 8% and kcat/KM = 2% of the wild type values for the transamination of aspartate and alpha-ketoglutarate. The affinity of the enzyme for the noncovalently bound inhibitor maleate is reduced 17-fold by the mutation, while only a 2.5-fold reduction is observed for alpha-methylaspartate, which forms a stable, covalent external aldimine. The high population of the quinonoid intermediate formed in the reaction of the wild type with beta-hydroxyaspartate is more than 75% diminished by the mutation. The values of the Y70F C alpha-H kinetic isotope effects for the aspartate reaction are larger than those of wild type (DV = 2.4 vs 1.52; D(V/K) = 2.5 vs 1.7). Conversely, the Y70F value of D(V/K) for the glutamate reaction is decreased compared to wild type (1.75 vs 2.5). These results, combined with previous studies of Lys258 mutants, eliminate Tyr70 as an essential component of the catalytic apparatus, with the caveat that the functionally of the deleted hydroxyl group is possibly replaced by a water molecule.
Aspartate Aminotransferases↗
Kinetics and equilibria for the reactions of coenzymes with wild type and the Y70F mutant of Escherichia coli aspartate aminotransferase.
The Y70F mutant of aspartate aminotransferase has reduced affinity for coenzymes compared to the wild type. The equilibrium dissociation constants for pyridoxamine phosphate (PMP) holoenzymes, KPMPdiss, were determined from the association and dissociation rate constants to be 1.3 nM and 30 nM for the wild type and mutant, respectively. This increase in KPMPdiss for Y70F is due to a 27-fold increase in the dissociation rate constant. Pyridoxal phosphate (PLP) association kinetics are complex, with three kinetic processes detectable for wild type and two for Y70F. A directly determined, accurate value of KPLPdiss for wild type enzyme has been difficult to obtain because of the low value of this constant. The values of KPLPdiss for the holoenzymes were determined indirectly through the measured values for KPMPdiss, glutamate-alpha-ketoglutarate half-reaction equilibrium constants, and the equilibrium constant for the transamination of PLP by glutamate catalyzed by Y70F. The values of KPLPdiss obtained by this procedure are 0.4 pM for wild type and 40 pM for Y70F. The increases in KPMPdiss and KPLPdiss for Y70F correspond to delta delta G values of 1.9 and 2.7 kcal/mol, respectively, and are directly attributed to the loss of the hydrogen bond from the phenolic hydroxyl group of Tyr70 to the coenzyme phosphate. The delta G for association of PLP with wild type enzyme is 4.7 kcal/mol more favorable than that for PMP.
Apoenzymes↗
The tyrosine-225 to phenylalanine mutation of Escherichia coli aspartate aminotransferase results in an alkaline transition in the spectrophotometric and kinetic pKa values and reduced values of both kcat and Km.
Tyrosine-225 is hydrogen-bonded to the 3'-hydroxyl group of pyridoxal 5'-phosphate in the active site of aspartate aminotransferase. Replacement of this residue with phenylalanine (Y225F) results in a shift in the acidic limb of the pKa of the kcat/KAsp vs pH profile from 7.1 (wild-type) to 8.4 (mutant). The change in the kinetic pKa is mirrored by a similar shift in the spectrophotometrically determined pKa of the protonated internal aldimine. Thus, a major role of tyrosine-225 is to provide a hydrogen bond that stabilizes the reactive unprotonated form of the internal aldimine in the neutral pH range. The Km value for L-aspartate and the dissociation constant for alpha-methyl-DL-aspartate are respectively 20- and 37-fold lower in the mutant than in the wild-type enzyme, while the dissociation constant for maleate is much less perturbed. These results are interpreted in terms of competition between the Tyr225 hydroxyl group and the substrate or quasi-substrate amino group for the coenzyme. The value of kcat in Y225F is 450-fold less than the corresponding rate constant in wild type. The increased affinity of the mutant enzyme for substrates, combined with the lack of discrimination against deuterium in the C alpha position of L-aspartate in Y225F-catalyzed transamination [Kirsch, J. F., Toney, M. D., & Goldberg, J. M. (1990) in Protein and Pharmaceutical Engineering (Craik, C. S., Fletterick, R., Matthews, C. R., & Wells, J., Eds.) pp 105-118, Wiley-Liss, New York], suggests that the rate-determining step in the mutant is hydrolysis of the ketimine intermediate rather than C alpha-H abstraction which is partially rate-determining in wild type.
Aspartate Aminotransferases↗
Ancestral lysozymes reconstructed, neutrality tested, and thermostability linked to hydrocarbon packing.
The controversy surrounding the idea that neutral mutations dominate protein evolution is attributable in part to the inadequacy of the tools available to evolutionary investigators. With a few exceptions, most investigations into the force driving protein evolution have relied on indirect criteria for distinguishing neutral and non-neutral variants. To investigate a particular pathway of molecular evolution, we have reconstructed by site-directed mutagenesis likely ancestral variants of the lysozymes of modern game birds (order Galliformes), tested their activity and thermostability and determined their three-dimensional structure. We focused on amino acids at three positions that are occupied in all known game birds either by the triplet Thr 40, Ile 55, Ser 91, or by the triplet Ser 40, Val 55, Thr 91. We have synthesized proteins representing intermediates along the possible three-step evolutionary pathways between these triplets. Although all of these are active and stable, none of these intermediates is found in known lysozymes. A comparison of the structures and thermostabilities of the variants reveals a linear correlation between the side-chain volume of the triplet and the thermostability of the protein. Each pathway connecting the two extant triplet sequences includes a variant with a thermostability outside the range of the extant proteins. This observation is consistent with a non-neutral evolutionary pathway. The existence of variants that are more stable than the extant proteins suggests that selection for maximum thermostability may not have been an important factor in the evolution of this enzyme.
Amino Acid Sequence↗
Sequential protection-modification method for selective sulfhydryl group derivatization in proteins having more than one cysteine.
The method of Smith and Hartman [J. Biol. Chem., 263, 4921-4925 (1988)] for introducing the non-natural lysine analog, S-(2-aminoethyl)cysteine, into specific sites in proteins by alkylation of a genetically introduced cysteine with 2-bromoethylamine has been generalized to be applicable to proteins containing one or more endogenous cysteines. The target cysteine residue introduced at the active site of aspartate aminotransferase is protected by bound cofactor. The enzyme is partially unfolded in low concentrations of urea, and the non-active site cysteine residues derivatized by a reversible thiol protecting reagent. The active site cysteine is then exposed and alkylated in 6 M urea. Enzyme activity is regenerated by removal of the thiol protecting groups and refolding of the protein.
Alkylating Agents↗
Brønsted analysis of enzymatic proton transfer reactions through site-directed mutagenesis.
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Animals↗
Estimation of free energy barriers in the cytoplasmic and mitochondrial aspartate aminotransferase reactions probed by hydrogen-exchange kinetics of C alpha-labeled amino acids with solvent.
The existence of the postulated quinonoid intermediate in the cytoplasmic aspartate amino-transferase catalyzed transamination of aspartate to oxaloacetate was probed by determining the extent of transfer of tritium from the C alpha position of tritiated L-aspartate to pyridoxamine 5'-phosphate in single turnover experiments in which washout from the back-reaction was obviated by product trapping. The maximum amount of transferred tritium observed was 0.7%, consistent either with a mechanism in which a fraction of the net transamination reaction proceeds through a quinonoid intermediate or with a mechanism in which this intermediate is formed off the main reaction pathway. It is shown that transfer of labeled hydrogen from the amino acid to cofactor cannot be used to differentiate a stepwise from a concerted transamination mechanism. The amount of tritium transferred is a function of the rate constant for torsional equilibration about the epsilon-amino group of Lys-258, the presumptive abstractor of the C alpha proton; the relative rate constants for hydrogen exchange with solvent versus cofactor protonation; and the tritium isotope effect on this ratio. The free energy barriers facing the covalent intermediate between aldimine and keto acid product (i.e., ketimine and possibly quinonoid) were evaluated relatively by comparing the rates of C alpha-hydrogen exchange in starting amino acid with the rates of keto acid formation. The value of theta (= kexge/kprod) was found to be 2.6 for the reaction of cytoplasmic isozyme with aspartate and ca. 0.5 for that of the mitochondrial form with glutamate.
Animals↗
Aspartate aminotransferase catalyzed oxygen exchange with solvent from oxygen-18-enriched alpha-ketoglutarate: evidence for slow exchange of enzyme-bound water.
Partitioning of the ketimine (or ketimine + quinonoid) intermediate(s) in the mitochondrial aspartate aminotransferase reactions was investigated by following the rates of loss of 18O from carbonyl-18O-enriched alpha-ketoglutarate together with the rate of L-glutamate formation. The ratio of these rate constants was found to equal 1 at 10 degrees C, implying that the above intermediate(s) face(s) equal barriers with respect to the forward and reverse reactions. This partition ratio of 1 together with that measured from the alpha-amino acid side of the reaction [Julin, D.A., Wiesinger, H., Toney, M. D., & Kirsch, J.F. (1989) Biochemistry (preceding paper in this issue)] suggests that the rate constant for exchange of alpha-ketoglutarate-derived H2(18)O from the ketimine (or ketimine + quinonoid) form(s) of the enzyme with solvent is comparable with that for kcat.
Animals↗
Kinetic isotope effect studies on aspartate aminotransferase: evidence for a concerted 1,3 prototropic shift mechanism for the cytoplasmic isozyme and L-aspartate and dichotomy in mechanism.
The C alpha primary hydrogen kinetic isotope effects (C alpha-KIEs) for the reaction of the cytoplasmic isozyme of aspartate aminotransferase (cAATase) with [alpha-2H]-L-aspartate are small and only slightly affected by deuterium oxide solvent (DV = 1.43 +/- 0.03 and DV/KAsp = 1.36 +/- 0.04 in H2O; DV = 1.44 +/- 0.01 and DV/KAsp = 1.61 +/- 0.06 in D2O). The D2O solvent KIEs (SKIEs) are somewhat larger and are essentially independent of deuterium at C alpha (D2OV = 2.21 +/- 0.07 and D2OV/KAsp = 1.70 +/- 0.03 with [alpha-1H]-L-aspartate; D2OV = 2.34 +/- 0.12 and D2OV/KAsp = 1.82 +/- 0.06 with [alpha-2H]-L- aspartate). The C alpha-KIEs on V and on V/KAsp are independent of pH from pH 5.0 to pH 10.0. These results support a rate-determining concerted 1,3 prototropic shift mechanism by the multiple KIE criteria [Hermes, J. D., Roeske, C. A., O'Leary, M. H., & Cleland, W. W. (1982) Biochemistry 21, 5106]. The large C alpha-KIEs for the reaction of mitochondrial AATase (mAATase) with L-glutamate (DV = 1.88 +/- 0.13 and DV/KGlu = 3.80 +/- 0.43 in H2O; DV = 1.57 +/- 0.05 and DV/KGlu = 4.21 +/- 0.19 in D2O) coupled with the relatively small SKIEs (D2OV = 1.58 +/- 0.04 and D2OV/KGlu = 1.25 +/- 0.05 with [alpha-1H]-L-glutamate; D2OV = 1.46 +/- 0.06 and D2OV/KGlu = 1.16 +/- 0.05 with [alpha-2H]-L-glutamate) are most consistent with a two-step mechanism for the 1,3 prototropic shift for this isozyme-substrate pair.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Direct Brønsted analysis of the restoration of activity to a mutant enzyme by exogenous amines.
A true Brønsted analysis of proton transfer in an enzyme mechanism is made possible by the chemical rescue of an inactive mutant of aspartate aminotransferase, where the endogenous general base, Lys258, is replaced with Ala by site-directed mutagenesis. Catalytic activity is restored to this inactive mutant by exogenous amines. The eleven amines studied generate a Brønsted correlation with beta of 0.4 for the transamination of cysteine sulfinate, when steric effects are included in the regression analysis. Localized mutagenesis thus allows the classical Brønsted analysis of transition-state structure to be applied to enzyme-catalyzed reactions.
Amines↗
Isolation of a Staphylococcus aureus beta-lactamase-dicloxacillin complex and kinetic studies on the reactivation of the enzyme.
Exposure of the beta-lactamase from Staphylococcus aureus to the slowly reacting substrates cloxacillin or dicloxacillin results in time-dependent inactivation of the enzyme. Methods for the rapid separation of a beta-lactamase-dicloxacillin complex from excess inhibitor, using centrifuged columns of Sephadex G-25 or DEAE-Sephadex G-25, are described. The enzyme-dicloxacillin complex releases active enzyme, with specific activity identical to that of untreated enzyme, after storage at pH 7.5 at 15 degrees C. Full reactivation was accompanied by the release of 0.8 eq of hydrolyzed dicloxacillin. The complex is stable for up to 40 h when stored at pH 3 at 4 degrees C. The reactivation process, which occurs with first-order kinetics at 15 degrees C and pH values between 4 and 8, displays a pH dependence with apparent pKa's of 4.6 and 8.5, and a limiting value of the reactivation rate constant of 0.022 min-1. Deviation from first-order kinetics at pH 9 is consistent with a competing irreversible inactivation of the enzyme at that pH. This behavior differs substantially from that of the similarly inactivated beta-lactamase I from Bacillus cereus, whose rate of reactivation is independent of pH, but which undergoes irreversible denaturation at acidic pH [A. L. Fink, K. M. Behner, and A. K. Tan (1987) Biochemistry 26, 4248-4258]. Addition of hydroxylamine to the S. aureus beta-lactamase-dicloxacillin, complex stimulates the rate of reactivation by a maximum of 35%. This effect is hyperbolically dependent on the concentration of hydroxylamine with half-maximal stimulation at 2.8 mM. The Km for ampicillin hydrolysis catalyzed by the partially reactivated enzyme is identical to that measured for catalysis by the untreated enzyme. We discuss our observations in relation to models for the transient inhibition process.
Dicloxacillin↗