The kinetic mechanism of wild-type and mutant mouse dihydrofolate reductases

A kinetic mechanism is presented for mouse dihydrofolate reductase that predicts all the steady-state parameters and full time-course kinetics. This mechanism was derived from association and dissociation rate constants and pre-steady-state transients by using stopped-flow fluorescence and absorbance measurements. The major features of this kinetic mechanism are as follows: (1) the two native enzyme conformers, E1 and E2, bind ligands with varying affinities although only one conformer, E1, can support catalysis in the forward direction, (2) tetrahydrofolate dissociation is the rate-limiting step under steady-state turnover at low pH, and (3) the pH-independent rate of hydride transfer from NADPH to dihydrofolate is fast (khyd = 9000 s-1) and favorable (Keq = 100). The overall mechanism is similar in form to the Escherichia coli kinetic scheme (Fierke et al., 1987), although several differences are observed: (1) substrates and products predominantly bind the same form of the E. coli enzyme, and (2) the hydride transfer rate from NADPH to either folate or dihydrofolate is considerably faster for the mouse enzyme. The role of Glu-30 (Asp-27 in E. coli) in mouse DHFR has also been examined by using site-directed mutagenesis as a potential source of these differences. While aspartic acid is strictly conserved in all bacterial DHFRs, glutamic acid is conserved in all known eucaryotes. The two major effects of substituting Asp for Glu-30 in the mouse enzyme are (1) a decreased rate of folate reduction and (2) an increased rate of hydride transfer from NADPH to dihydrofolate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of enzyme and ligand protonation on the binding of folates to recombinant human dihydrofolate reductase: implications for the evolution of eukaryotic enzyme efficiency.

There is marked pH dependence of the rate constant (koff) for tetrahydrofolate (H4folate) dissociation from its ternary complex with human dihydrofolate reductase (hDHFR) and NADPH. Similar pH dependence of H4folate dissociation from the ternary complex of a variant of hDHFR with the substitution Phe31----Leu (F31L hDHFR) causes this dissociation to become rate limiting in the enzyme mechanism at pH approximately 5, and this accounts for the marked decrease in kcat for this variant as the pH is decreased from 7 to 5. This decreased kcat at low pH is not seen for most DHFRs. koff for dissociation of folate, dihydrofolate (H2folate), and H4folate from their binary complexes with hDHFR is similarly pH dependent. For all the complexes examined, the pH dependence of koff in the range pH 5-7 is well described by a pKa of about 6.2 and must be due to ionization of a group on the enzyme. In the higher pH range (7-10), koff increases further as the pH is raised, and this relation is governed by a second pKa which is close to the pKa for ionization of the amide group (HN3-C4O) of the respective ligands. Thus, ionization of the ligand amide group also increases koff. Evidence is presented that the dependence of pH on koff for hDHFR accounts for the shape of the kcat versus pH curve for both hDHFR as well as its F31L variant and contributes to the higher efficiency of hDHFR compared with bacterial DHFR.     

Construction and evaluation of the kinetic scheme associated with dihydrofolate reductase from Escherichia coli

A kinetic scheme is presented for Escherichia coli dihydrofolate reductase that predicts steady-state kinetic parameters and full time course kinetics under a variety of substrate concentrations and pHs. This scheme was derived from measuring association and dissociation rate constants and pre-steady-state transients by using stopped-flow fluorescence and absorbance spectroscopy. The binding kinetics suggest that during steady-state turnover product dissociation follows a specific, preferred pathway in which tetrahydrofolate (H4F) dissociation occurs after NADPH replaces NADP+ in the ternary complex. This step, H4F dissociation from the E X NADPH X H4F ternary complex, is proposed to be the rate-limiting step for steady-state turnover at low pH because koff = VM. The rate constant for hydride transfer from NADPH to dihydrofolate (H2F), measured by pre-steady-state transients, has a deuterium isotope effect of 3 and is rapid, khyd = 950 s-1, essentially irreversible, Keq = 1700, and pH dependent, pKa = 6.5, reflecting ionization of a single group in the active site. This scheme accounts for the apparent pKa = 8.4 observed in the steady state as due to a change in the rate-determining step from product release at low pH to hydride transfer above pH 8.4. This kinetic scheme is a necessary background to analyze the effects of single amino acid substitutions on individual rate constants.     

Impact on catalysis of secondary structural manipulation of the alpha C-helix of Escherichia coli dihydrofolate reductase

The alpha C-helix of Escherichia coli dihydrofolate reductase has been converted to its counterpart in Lactobacillus casei by a triple mutation in the helix (H45R, W47Y, and I50F). These changes result in a 2-fold increase in the steady-state reaction rate (kcat = 26 s-1) that is limited by an increased off rate for the release of tetrahydrofolate (koff = 40 s-1 versus 12 s-1). On the other hand the mutant protein exhibits a 10-fold increase in the KM value (6.8 microM) for dihydrofolate and a 10-fold decrease in the rate of hydride transfer (85 s-1) from NADPH to dihydrofolate. The elevated rate of tetrahydrofolate release upon the rebinding of NADPH, a characteristic of the wild-type enzyme-catalyzed reaction, is diminished. The intrinsic pKa (6.4) of the mutant enzyme binary complex with NADPH is similar to that of the wild type, but the pKa of the ternary complex is increased to 7.3, about on pH unit higher than the wild-type value. Further mutagenesis (G51P and an insertion of K52) was conducted to incorporate a hairpin turn unique to the C-terminus of the alpha C-helix of the L. casei enzyme in order to adjust a possible dislocation of the new helix. The resultant pentamutant enzyme shows restoration of many of the kinetic parameters, such as kcat (12 s-1), KM (1.1 microM for dihydrofolate), and khyd (526 s-1), to the wild-type values. The synergism in the product release is also largely restored. A substrate-induced conformational change responsible for the fine tuning of the catalytic process was found to be associated with the newly installed hairpin structure. The Asp27 residue of the mutant enzyme was found to be reprotonated before tetrahydrofolate release.     

Probing the functional role of phenylalanine-31 of Escherichia coli dihydrofolate reductase by site-directed mutagenesis

The role of Phe-31 of Escherichia coli dihydrofolate reductase in binding and catalysis was probed by amino acid substitution. Phe-31, a strictly conserved residue located in a hydrophobic pocket and interacting with the pteroyl moiety of dihydrofolate (H2F), was replaced by Tyr and Val. The kinetic behavior of the mutant enzymes in general is similar to that of the wild type. The rate-limiting step for both mutant enzymes is the release of tetrahydrofolate (H4F) from the E X NADPH X H4F ternary complex as determined for the wild type. The 2-fold increase in V for the two mutant enzymes arises from faster dissociation of H4F from the enzyme-product complex. The quantitative effect of these mutations is to decrease the rate of hydride transfer, although not to the extent that this step becomes partially rate limiting, but to accelerate the dissociation rates of tetrahydrofolate from product complexes so that the opposing effects are nearly compensating.     

Hydrophobic interactions via mutants of Escherichia coli dihydrofolate reductase: separation of binding and catalysis

The strictly conserved residue leucine-54 of Escherichia coli dihydrofolate reductase forms part of the hydrophobic wall which binds the p-aminobenzoyl side chain of dihydrofolate. In addition to the previously reported glycine-54 mutant, isoleucine-54 and asparagine-54 substitutions have been constructed and characterized with regard to their effects on binding and catalysis. NADP+ and NADPH binding is virtually unaffected with the exception of a 15-fold decrease in NADPH dissociation from the Gly-54 mutant. The synergistic effect of NADPH on tetrahydrofolate dissociation seen in the wild-type enzyme is lost in the isoleucine-54 mutant: little acceleration is seen in tetrahydrofolate dissociation when cofactor is bound, and there is no discrimination between reduced and oxidized cofactor. The dissociation constants for dihydrofolate and methotrexate increase in the order Leu less than Ile less than Asn less than Gly, varying by a maximum factor of 1700 for dihydrofolate and 6300 for methotrexate. Despite these large changes in binding affinity, the hydride transfer rate of 950 s-1 in the wild-type enzyme is decreased by a constant factor of ca. 30 (2 kcal/mol) regardless of the mutant. Thus, the contributions of residue 54 to binding and catalysis appear to have been separated.     

Role of the conserved active site residue tryptophan-24 of human dihydrofolate reductase as revealed by mutagenesis

The active sites of all bacterial and vertebrate dihydrofolate reductases that have been examined have a tryptophan residue near the binding sites for NADPH and dihydrofolate. In cases where the three-dimensional structure has been determined by X-ray crystallography, this conserved tryptophan residue makes hydrophobic and van der Waals interactions with the nicotinamide moiety of bound NADPH, and its indole nitrogen interacts with the C4 oxygen of bound folate through a bridge provided by a bound water molecule. We have addressed the question of why even the very conservative replacement of this tryptophan by phenylalanine does not seem to occur naturally. Human dihydrofolate reductase with this replacement of tryptophan by phenylalanine has increased rate constants for dissociation of substrates and products and a considerably decreased rate of hydride transfer. These cause some changes in steady-state kinetic behavior, including substantial increases in Michaelis constants for NADPH and dihydrofolate, but there is also a 3-fold increase in kcat. The branched mechanistic pathway for this enzyme has been completely defined and is sufficiently different from that of wild-type enzyme to cause changes in some transient-state kinetics. The most critical changes resulting from the amino acid substitution appear to be a 50% decrease in stability and a decrease in efficiency from 69% to 21% under intracellular conditions.     

A kinetic study of wild-type and mutant dihydrofolate reductases from Lactobacillus casei

A kinetic scheme is presented for Lactobacillus casei dihydrofolate reductase that predicts steady-state kinetic parameters. This scheme was derived from measuring association and dissociation rate constants and pre-steady-state transients by using stopped-flow fluorescence and absorbance spectroscopy. Two major features of this kinetic scheme are the following: (i) product dissociation is the rate-limiting step for steady-state turnover at low pH and follows a specific, preferred pathway in which tetrahydrofolate (H4F) dissociation occurs after NADPH replaces NADP+ in the ternary complex; (ii) the rate constant for hydride transfer from NADPH to dihydrofolate (H2F) is rapid (khyd = 430 s-1), favorable (Keq = 290), and pH dependent (pKa = 6.0), reflecting ionization of a single group. Not only is this scheme identical in form with the Escherichia coli kinetic scheme [Fierke et al. (1987) Biochemistry 26, 4085] but moreover none of the rate constants vary by more than 40-fold despite there being less than 30% amino acid homology between the two enzymes. This similarity is consistent with their overall structural congruence. The role of Trp-21 of L. casei dihydrofolate reductase in binding and catalysis was probed by amino acid substitution. Trp-21, a strictly conserved residue near both the folate and coenzyme binding sites, was replaced by leucine. Two major effects of this substitution are on (i) the rate constant for hydride transfer which decreases 100-fold, becoming the rate-limiting step in steady-state turnover, and (ii) the affinities for NADPH and NADP+ which decrease by approximately 3.5 and approximately 0.5 kcal mol-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Hydride transfer by dihydrofolate reductase. Causes and consequences of the wide range of rates exhibited by bacterial and vertebrate enzymes

Transient and steady-state kinetics have been examined for dihydrofolate reductase (DHFR) from a number of sources. Rates of hydride transfer at pH 7.65 cover a wide range, from 7 s-1 for DHFR from a strain of Lactobacillus casei (LCDHFR1) to 3000 s-1 for recombinant human DHFR (rHDHFR). In all cases as the pH is increased from 7 to 10, Vmax for the steady-state reaction decreases, and DVmax, the primary isotope effect, increases. This indicates a decrease in the rate of hydride transfer with increasing pH. The cross-over points, at which rates of product release and hydride transfer become equal, were calculated to occur at DVmax = 2.34. The higher the rate of hydride transfer at pH 7.65, the higher the pH of the cross-over point. For LCDHFR1 the low rate of hydride transfer results in this process being partially rate-limiting for the steady-state reaction even at pH 5, with a cross-over point at about pH 7. At pH 7.65 the burst phase associated with the initial conversion of enzyme-bound substrates to enzyme-bound products has an isotope effect of 3 or higher for LCDHFR and for DHFR from Escherichia coli (ECDHFR). In contrast, the vertebrate DHFRs (bovine, BDHFR; chicken, CDHFR; and rHDHFR) exhibit a burst of product formation which is only partially limited by hydride transfer at this pH (Dkb: 2.3, 2.2, and 2.1, respectively). An obligatory isomerization of the ternary substrate complex or of the ternary product complex is postulated to be partially rate-limiting for the vertebrate enzymes. At pH 5 LCDHFR1 and ECDHFR also exhibit evidence of such a rate-limiting obligatory conformational transition of the substrate or product ternary complex.     

Complementary perturbation of the kinetic mechanism and catalytic effectiveness of dihydrofolate reductase by side-chain interchange.

The variable residue Leu-28 of Escherichia coli dihydrofolate reductase (DHFR) and the corresponding residue Phe-31 in murine DHFR were interchanged, and the impact on catalysis was evaluated by steady-state and pre-steady-state analysis. The E. coli L28F mutant increased the pH-independent kcat from 11 to 50 s-1 but had little effect on Km(H2F). An increase in the rate constant for dissociation of H4F from E.H4F.NH (from 12 to 80 s-1) was found to be largely responsible for the increase in kcat. Unexpectedly, the rate constant for hydride transfer increased from 950 to 4000 s-1 with little perturbation of NADPH and NADP+ binding to E. Consequently, the flux efficiency of the E. coli L28F mutant rose from 15% to 48% and suggests a role in genetic selection for this variable side chain. The murine F31L mutant decreased the pH-independent kcat from 28 to 4.8 s-1 but had little effect on Km(H2F). A decrease in the rate constant for dissociation of H4F from E.H4F.NH (from 40 to 22 s-1) and E.H4F (from 15 to 0.4 s-1) was found to be mainly responsible for the decrease in kcat. The rate constant for hydride transfer decreased from 9000 to 5000 s-1 with minor perturbation of NADPH binding. Thus, the free energy differences along the kinetic pathway were generally similar in magnitude but opposite in direction to those incurred by the E. coli L28F mutant. This conclusion implies that DHFR hydrophobic active-site side chains impart their characteristics individually and not collectively.     

Investigation of the functional role of tryptophan-22 in Escherichia coli dihydrofolate reductase by site-directed mutagenesis

We have applied site-directed mutagenesis methods to change the conserved tryptophan-22 in the substrate binding site of Escherichia coli dihydrofolate reductase to phenylalanine (W22F) and histidine (W22H). The crystal structure of the W22F mutant in a binary complex with the inhibitor methotrexate has been refined at 1.9-A resolution. The W22F difference Fourier map and least-squares refinement show that structural effects of the mutation are confined to the immediate vicinity of position 22 and include an unanticipated 0.4-A movement of the methionine-20 side chain. A conserved bound water-403, suspected to play a role in the protonation of substrate DHF, has not been displaced by the mutation despite the loss of a hydrogen bond with tryptophan-22. Steady-state kinetics, stopped-flow kinetics, and primary isotope effects indicate that both mutations increase the rate of product tetrahydrofolate release, the rate-limiting step in the case of the wild-type enzyme, while slowing the rate of hydride transfer to the point where it now becomes at least partially rate determining. Steady-state kinetics show that below pH 6.8, kcat is elevated by up to 5-fold in the W22F mutant as compared with the wild-type enzyme, although kcat/Km(dihydrofolate) is lower throughout the observed pH range. For the W22H mutant, both kcat and kcat/Km(dihydrofolate) are substantially lower than the corresponding wild-type values. While both mutations weaken dihydrofolate binding, cofactor NADPH binding is not significantly altered. Fitting of the kinetic pH profiles to a general protonation scheme suggests that the proton affinity of dihydrofolate may be enhanced upon binding to the enzyme. We suggest that the function of tryptophan-22 may be to properly position the side chain of methionine-20 with respect to N5 of the substrate dihydrofolate.     

Subsite interactions of ribonuclease T1: viscosity effects indicate that the rate-limiting step of GpN transesterification depends on the nature of N

We report on the effect of the viscogenic agents glycerol and ficoll on the RNase T1 catalyzed turnover of GpA, GpC, GpU, and Torula yeast RNA. For wild-type enzyme, we find that the kcat/Km values for the transesterification of GpC and GpA as well as for the cleavage of RNA are inversely proportional to the relative viscosity of glycerol-containing buffers; no such effect is observed for the conversion of GpU to cGMP and U. The second-order rate constants for His40Ala and Glu46Ala RNase T1, two mutants with a drastically reduced kcat/km ratio, are independent of the microviscosity, indicating that glycerol does not affect the intrinsic kinetic parameters. Consistent with the notion that molecular diffusion rates are unaffected by polymeric viscogens, addition of ficoll has no effect on the kcat/Km for GpC transesterification by wild-type enzyme. The data indicate that the second-order rate constants for GpC, GpA, and Torula yeast RNA are at least partly limited by the diffusion-controlled association rate of substrate and active site; RNase T1 obeys Briggs-Haldane kinetics for these substrates (Km greater than Ks). Calculations suggest that the equilibrium dissociation constants (Ks) for the various GpN-wild-type enzyme complexes are virtually independent of N whereas the measured kcat values follow the order GpC greater than GpA greater than GpU. This is also revealed by the steady-state kinetic parameters of Tyr38Phe and His40Ala RNase T1, two mutants that follow simple Michaelis-Menten kinetics because of a dramatically reduced kcat value (i.e., Km = Ks).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Effects of conversion of phenylalanine-31 to leucine on the function of human dihydrofolate reductase

Oligonucleotide-directed, site-specific mutagenesis was used to convert phenylalanine-31 of human recombinant dihydrofolate reductase (DHFR) to leucine. This substitution was of interest in view of earlier chemical modification studies (Kumar et al., 1981) and structural studies based on X-ray crystallographic data (Matthews et al., 1985a,b) which had implicated the corresponding residue in chicken liver DHFR, Tyr-31, in the binding of dihydrofolate. Furthermore, this particular substitution allowed testing of the significance of protein sequence differences between mammalian and bacterial reductases at this position with regard to the species selectivity of trimethoprim. Both wild-type (WT) and mutant (F31L) enzymes were expressed and purified by using a heterologous expression system previously described (Prendergast et al., 1988). Values of the inhibition constants (Ki values) for trimethoprim were 1.00 and 1.08 microM for WT and F31L, respectively. Thus, the presence of phenylalanine at position 31 in human dihydrofolate reductase does not contribute to the species selectivity of trimethoprim. The Km values for nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) and dihydrofolate were elevated 10.8-fold and 9.4-fold, respectively, for the mutant enzyme, whereas the Vmax increased only 1.8-fold. Equilibrium dissociation constants (KD values) were obtained for the binding of NADPH and dihydrofolate in binary complexes with each enzyme. The KD for NADPH is similar in both WT and F31L, whereas the KD for dihydrofolate is 43-fold lower in F31L. Values for dihydrofolate association rate constants (kon) with enzyme and enzyme-NADPH complexes were measured by stopped-flow techniques.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of the formation and isomerization of methotrexate complexes of recombinant human dihydrofolate reductase

The kinetics of inhibitor binding to highly purified recombinant human dihydrofolate reductase (rHDHFR) have been examined. Methotrexate (MTX) binds rapidly (kon = 1.0 x 10(8) M-1 s-1) and tightly (koff/kon = 210 pM) to the preformed complex of rHDHFR with NADPH. The initial association reaction between rHDHFR.NADPH and MTX is followed by an isomerization of the resulting complex (kiso = 0.4 s-1) leading to a new conformer in which MTX is bound even more tightly (Ki = 3.4 pM). Similar results have been obtained with a major metabolite of MTX having four additional glutamate residues for which Ki = 1.4 pM. 7-HydroxyMTX, another major metabolite of MTX, is a weak inhibitor of rHDHFR (Ki = 8.9 nM), and a polyglutamate form of this metabolite is an equally weak inhibitor (Ki = 9.9 nM), so that the addition of glutamate residues to MTX or 7-hydroxyMTX has little effect on their binding. It follows that the significance of MTX polyglutamate formation relates to other roles such as increasing the cytotoxicity of MTX by prolonging intracellular retention of the drug. Another antifolate, trimethoprim, binds tightly to dihydrofolate reductases from bacterial sources, but weakly to rHDHFR in the ternary complex (KD = 0.5 microM). Although the association step is rapid (kon = 0.4 x 10(8) M-1 s-1), the dissociation rate is also rapid (koff = 15 s-1). Furthermore, there is no isomerization of the ternary complex of trimethoprim with rHDHFR, in contrast to the known isomerization of complexes of trimethoprim with bacterial dihydrofolate reductases.     

Vibrational structure of dihydrofolate bound to R67 dihydrofolate reductase.

R67 is a Type II dihydrofolate reductase (DHFR) that catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate by facilitating the addition of a proton to N5 of DHF and the transfer of a hydride ion from NADPH to C6. Because this enzyme is a plasmid-encoded DHFR from trimethoprim-resistant bacteria, extensive studies on R67 with various methods have been performed to elucidate its reaction mechanism. Here, Raman difference measurements, conducted on the ternary complex of R67.NADP(+).DHF believed to be an accurate mimic of the productive DHFR.NADPH.DHF complex, show that the pK(a) of N5 in the complex is less than 4. This is in clear contrast to the behavior observed in Escherichia coli DHFR, a substantially more efficient enzyme, where the pK(a) of bound DHF at N5 is increased to 6.5 compared with its solution value of 2.6. A comparison of the ternary complexes in R67 and E. coli DHFRs suggests that enzymic raising of the pK(a) at N5 can significantly increase the catalytic efficiency of the hydride transfer step. However, R67 shows that even without such a strategy an effective DHFR can still be designed.     

(S)-Mandelate dehydrogenase from Pseudomonas putida: mechanistic studies with alternate substrates and pH and kinetic isotope effects

(S)-Mandelate dehydrogenase from Pseudomonas putida, a member of the flavin mononucleotide-dependent alpha-hydroxy acid oxidase/dehydrogenase family, oxidizes (S)-mandelate to benzoylformate. The enzyme was purified with a carboxy-terminal histidine tag. Steady-state kinetic parameters indicate that it preferentially binds large substrates. A good correlation was obtained between the kcat, the substrate kinetic isotope effect (KIE), and the pKa of the substrate alpha-proton. The kcat decreased and the KIE increased for substrates whose alpha-protons have pKas higher than that of mandelate. These results support a mechanism involving a carbanion intermediate but are difficult to reconcile with one involving a direct hydride transfer. pH effects on steady-state parameters were determined with (S)-mandelate and a slow substrate, (R,S)-3-phenyllactate. The kcat/Km pH profile shows that two groups with apparent pKas of 5.5 and 8.9 in the free enzyme are important for activity. These pKas are shifted to 5.1 and 9.6 on binding (S)-mandelate, as shown in the kcat pH profile. The pH dependence of the KIEs suggests that the residues with these pKas are involved in the alpha-carbon-hydrogen bond-breaking step. pH dependencies of the inhibition constants for competitive inhibitors identified these residues as histidine 274 and arginine 277. We propose that histidine 274 is the base that abstracts the substrate alpha-proton and arginine 277 is important for substrate binding as well as stabilization of the carbanion/enolate intermediate.     

Time-dependent 31P saturation transfer in the phosphoglucomutase reaction. Characterization of the spin system for the Cd(II) enzyme and evaluation of rate constants for the transfer process

Time-dependent 31P saturation-transfer studies were conducted with the Cd2+-activated form of muscle phosphoglucomutase to probe the origin of the 100-fold difference between its catalytic efficiency (in terms of kcat) and that of the more efficient Mg2+-activated enzyme. The present paper describes the equilibrium mixture of phosphoglucomutase and its substrate/product pair when the concentration of the Cd2+ enzyme approaches that of the substrate and how the nine-spin 31P NMR system provided by this mixture was treated. It shows that the presence of abortive complexes is not a significant factor in the reduced activity of the Cd2+ enzyme since the complex of the dephosphoenzyme and glucose 1,6-bisphosphate, which accounts for a large majority of the enzyme present at equilibrium, is catalytically competent. It also shows that rate constants for saturation transfer obtained at three different ratios of enzyme to free substrate are mutually compatible. These constants, which were measured at chemical equilibrium, can be used to provide a quantitative kinetic rationale for the reduced steady-state activity elicited by Cd2+ relative to Mg2+ [cf. Ray, W.J., Post, C.B., & Puvathingal, J.M. (1989) Biochemistry (following paper in this issue)]. They also provide minimal estimates of 350 and 150 s-1 for the rate constants describing (PO3-) transfer from the Cd2+ phosphoenzyme to the 6-position of bound glucose 1-phosphate and to the 1-position of bound glucose 6-phosphate, respectively. These minimal estimates are compared with analogous estimates for the Mg2+ and Li+ forms of the enzyme in the accompanying paper.