PubMed Health⌕ Search

Biomedical subjects

S J Benkovic

Publications and source records attributed to S J Benkovic.

At least 73 records · Page 4Linked to original sources
PubMed1998

Simultaneous formation of functional leading and lagging strand holoenzyme complexes on a small, defined DNA substrate.

The biochemical characterization of leading and lagging strand DNA synthesis by bacteriophage T4 replication proteins has been addressed utilizing a small, defined primer/template. The ATP hydrolysis activity of 44/62, the clamp loading complex responsible for holoenzyme assembly, was monitored during assembly of both the leading and lagging strand holoenzyme complex. The ATPase activity of 44/62 diminishes once a functional holoenzyme is assembled on both the leading and lagging strand. The assembly of the lagging strand holoenzyme is facilitated by several factors including biotinylated streptavidin blocks at the end of the fork strands, preassembly of the leading strand holoenzyme, and by the presence of the DNA primase with ribonucleoside triphosphates. The resultant minimal replicative complex consists of two holoenzymes and a primase nested on a model replication fork derived from a 62-mer template/34-mer primer/36-mer lagging strand in an apparent 2:2:1:1 ratio of 45 protein:polymerase:primase:forked DNA. The 44/62 protein complex does not remain associated with the complex. The primase alone slowly synthesizes pentaribonucleotides on the forked DNA when the lagging strand contains a nonannealed TTG initiation site with the rate of synthesis greatly stimulated by the addition of the 41 helicase. The addition of deoxy-NTPs to this complex results in leading strand synthesis, but extension of the synthesized RNA primer does not occur. DNA synthesis in both the leading and lagging strand directions is achieved, however, when a 6-mer DNA primer is annealed to the primase recognition site of the forked DNA substrate. A model is presented that describes how leading and lagging strand DNA synthesis might be coordinated as well as the associated molecular interactions of the replicative proteins.

Adenosine Triphosphatases↗
PubMed1998

Purification, characterization, and kinetic studies of a soluble Bacteroides fragilis metallo-beta-lactamase that provides multiple antibiotic resistance.

Resistance to multiple beta-lactam antibiotics traced to the expression of Zn(II) requiring metallo-beta-lactamases has emerged in clinical isolates of several bacterial strains including Bacteroides fragilis, a pathogen commonly found in suppurative/surgical infections. A soluble B. fragilis metallo-beta-lactamase has been purified to homogeneity from the cell growth medium after expression as a secretory protein in Escherichia coli. The enzyme requires two tightly bound Zn(II) ions for full activity, and the Zn(II) ions can be removed by EDTA from the enzyme. The apoenzyme is reactivated by stoichiometric amounts of Zn(II) and Co(II) ions. The Co(II)-substituted enzyme exhibits a UV-visible spectrum characterized by strong Co(II) d-d transitions at 510, 548, 615, and 635 nm and an EPR spectrum with g values of 5. 52, 4.25, and 2.01: features that serve as useful spectroscopic handles for the mechanistic studies of the enzyme. Although steady-state and transient-state kinetic studies of the soluble Zn(II) enzyme with nitrocefin as substrate found no ionizable groups with pKa values between 5.25 and 10.0 involved in catalysis, a kinetically significant proton transfer step in turnover was implicated by studies in deuterium oxide. These studies also detected the accumulation of an enzyme-bound intermediate and provide the basis for a minimal kinetic scheme describing metallo-beta-lactamase-catalyzed nitrocefin hydrolysis.

Bacteroides fragilis↗
PubMed1998

A pH-dependent stabilization of an active site loop observed from low and high pH crystal structures of mutant monomeric glycinamide ribonucleotide transformylase at 1.8 to 1.9 A.

A mutation in the dimer interface of Escherichia coli glycinamide ribonucleotide transformylase (GarTfase) disrupts the observed pH-dependent association of the wild-type enzyme, but has no observable effect on the enzyme activity. Here, we assess whether a pH effect on the enzyme's conformation is sufficient by itself to explain the pH-dependence of the GarTfase reaction. A pH-dependent conformational change is observed between two high-resolution crystal structures of the Glu70Ala mutant GarTfase at pH 3.5 (1.8 A) and 7.5 (1.9 A). Residues 110 to 131 in GarTfase undergo a transformation from a disordered loop at pH 3.5, where the enzyme is inactive, to an ordered loop-helix structure at pH 7.5, where the enzyme is active. The ordering of this flexible loop-helix has a direct effect on catalytic residues in the active site, binding of the folate cofactor and shielding of the active site from solvent. A main-chain carbonyl oxygen atom from Tyr115 in the ordered loop forms a hydrogen bond with His108, and thereby provides electronic and structural stabilization of this key active site residue. Kinetic data indicate that the pKa of His108 is in fact raised to 9. 2. The loop movement can be correlated with elevation of the His pKa, but with further stabilization, probably from Asp144, after the binding of folate cofactor. Leu118, also in the loop, becomes positioned near the p-amino benzoic acid binding site, providing additional hydrophobic interactions with the cofactor 10-formyl tetrahydrofolate. Thus, the pH-dependence of the enzyme activity appears to arise from local active site rearrangements and not from differences due to monomer-dimer association.

Binding Sites↗
PubMed1998

Evaluation of the kinetic mechanism of Escherichia coli glycinamide ribonucleotide transformylase.

A kinetic scheme is presented for Escherichia coli glycinamide ribonucleotide transformylase (GAR transformylase, EC 2.1.2.2) based on a steady-state and pre-steady-state kinetic analysis of the reaction in both directions employing stopped-flow absorbance and fluorescence spectroscopy. Steady-state parameters showed that kcat for the reverse direction is about 10 times lower than that for the forward direction although the Km values for formyl dideazafolate and dideazafolate or for glycinamide ribonucleotide and formyl glycinamide ribonucleotide are similar. No pre-steady-state transient was observed in either direction, and the single-turnover rate constant under saturating levels of substrates in each direction was found to be very close to the respective steady-state kcat value. This indicates that steps involving ternary complexes are rate-determining for steady-state turnover in each direction. By conducting the single-turnover reactions under various preincubation and mixing conditions, a random sequential kinetic mechanism was implicated in which the enzyme binds glycinamide ribonucleotide or formyl dideazafolate productively in no obligatory order. The collective data provided a quantitative kinetic scheme to serve as a basis for the analysis of mutations.

Bacterial Proteins↗
PubMed1998

Dissecting the order of bacteriophage T4 DNA polymerase holoenzyme assembly.

Most biological organisms rely upon a DNA polymerase holoenzyme for processive DNA replication. The bacteriophage T4 DNA polymerase holoenzyme is composed of the polymerase enzyme and a clamp protein (the 45 protein), which functions as a processivity factor by strengthening the interaction between DNA and the holoenzyme. The 45 protein must be loaded onto DNA by a clamp loader ATPase complex (the 44/62 complex). In this paper, the order of events leading to holoenzyme formation is investigated using a combination of rapid-quench and stopped-flow fluorescence spectroscopy kinetic methods. A rapid-quench strand displacement assay in which the order of holoenzyme component addition is varied provided data indicating that the rate-limiting step in holoenzyme assembly is associated with the clamp loading process. Pre-steady-state analysis of the clamp loader ATPase activity demonstrated that the four bound ATP molecules are hydrolyzed stepwise during the clamp loading process in groups of two. Clamp loading was examined with stopped-flow fluorescence spectroscopy from the perspective of the clamp itself, using a site-specific, fluorescently labeled 45 protein. A mechanism for T4 DNA polymerase holoenzyme assembly is proposed in which the 45 protein interacts with the 44/62 complex leading to the hydrolysis of 2 equiv of ATP, and upon contacting DNA, the remaining two ATP molecules bound to the 44/62 complex are hydrolyzed. Once all four ATP molecules are hydrolyzed, the 45 protein is poised on DNA for association with the polymerase to form the holoenzyme.

Adenosine Triphosphatases↗
PubMed1998

Deletion of a highly motional residue affects formation of the Michaelis complex for Escherichia coli dihydrofolate reductase.

Analysis of the dihydrofolate reductase (DHFR) complex with folate by two-dimensional heteronuclear (1H-15N) nuclear magnetic relaxation revealed that isolated residues exhibit diverse backbone fluctuations on the nanosecond to picosecond time scale [Epstein, D. M., Benkovic, S. J., and Wright, P. E. (1995) Biochemistry 34, 11037-11048]. These dynamical features may be significant in forming the Michaelis complex. Of these residues, glycine 121 displays large-amplitude backbone motions on the nanosecond time scale. This amino acid, strictly conserved for prokaryotic DHFRs, is located at the center of the betaF-betaG loop. To investigate the catalytic importance of this residue, we report the effects of Gly121 deletion and glycine insertion into the modified betaF-betaG loop. Relative to wild type, deletion of Gly121 dramatically decreases the rate of hydride transfer 550-fold and the strength of cofactor binding 20-fold for NADPH and 7-fold for NADP+. Furthermore, DeltaG121 DHFR requires conformational changes dependent on the initial binary complex to attain the Michaelis complex poised for hydride transfer. Surprisingly, the insertion mutants displayed a significant decrease in both substrate and cofactor binding. The introduction of glycine into the modified betaF-betaG loop, however, generally eliminated conformational changes required by DeltaG121 DHFR to attain the Michaelis complex. Taken together, these results suggest that the catalytic role for the betaF-betaG loop includes formation of liganded complexes and proper orientation of substrate and cofactor. Through a transient interaction with the Met20 loop, alterations of the betaF-betaG loop can orchestrate proximal and distal effects on binding and catalysis that implicate a variety of enzyme conformations participating in the catalytic cycle.

Escherichia coli↗
PubMed1998

Strength of an interloop hydrogen bond determines the kinetic pathway in catalysis by Escherichia coli dihydrofolate reductase.

On the basis of X-ray crystallographic data, Sawaya and Kraut proposed that Met20 loop conformational changes modulate ligand specificity observed in the catalytic cycle for Escherichia coli dihydrofolate reductase (DHFR) [Sawaya, M. R., and Kraut, J. (1997) Biochemistry 36, 586-603]. Interloop hydrogen bonds stabilize either a closed Met20 loop conformation observed in substrate complexes or an occluded Met20 loop conformation observed in product complexes, respectively. To test this model, we targeted a single hydrogen bond occurring exclusively in the closed Met20 loop conformation. Specifically, Asp122 in the betaF-betaG loop was independently substituted with asparagine, serine, and alanine-amino acids with decreasing abilities to hydrogen-bond. The kinetic analyses of the Asp122 mutants enabled the construction of kinetic schemes at pH 7.0 that demonstrate two striking features. First, a significant correlation exists between decreased binding of nicotinamide adenine dinucleotide phosphate, reduced (NADPH), and decreased hydride transfer rates resulting from these mutations. In other words, the interactions of Asp122 are along the reaction coordinate leading to the transition state. Second, substitutions for Asp122 alter the catalytic pathway preferred by wild-type DHFR under saturating conditions of substrate and cofactor. Overall, the steady-state rate contains contributions from the product off rates from the DHFR.5,6, 7,8-tetrahydrofolate (H4F) and DHFR.NADPH.H4F complexes and from the rate of hydride transfer. These mutational effects support the mechanistic model whereby interloop contacts regulate an equilibrium of Met20 loop conformations that, in turn, modulate ligand affinity and turnover.

Catalysis↗
PubMed1998

Effect of accessory proteins on T4 DNA polymerase replication fidelity.

The influence of replication accessory proteins on the fidelity of T4 DNA polymerase has been examined. Steady-state kinetic measurements showed that exonuclease-deficient T4 DNA polymerase, alone or with clamp loaders gp44/gp62 and polymerase clamp gp45, displays decreased binding affinity for incorrect as compared to correct dNTPs and a deceased kcat for misinsertion as compared to correct insertion. Kinetic constants were similar with and without accessory proteins, indicating that accessory proteins had little effect on misinsertion. They also had little effect on the Km value for extension of a T.T mismatch. However, the kcat value for T.T mismatch extension was fivefold higher in the presence of the clamp loader and clamp proteins. Thus, in the absence of proofreading, these accessory proteins may promote stable misincorporation. The kinetic analysis is supported by error rate determinations during gap-filling synthesis, which require both misinsertion and mispair extension. For some mispairs, the accuracy of exonuclease-deficient polymerase alone is similar to that in the presence of clamp loader, clamp and single-stranded DNA binding protein (gp32). However, exonuclease-deficient holoenzyme complex is actually less accurate than the polymerase alone for some base substitutions. We suggest that gp45 promotes extension of mismatches by tethering the polymerase to DNA, a process that may be relevant to replication past lesions or other blocks to DNA synthesis. The error rate for one-nucleotide deletions in homopolymeric runs was similar for the polymerase with or without its accessory proteins. This implies that strand misalignment errors arise during highly processive replication. Thus, either unpaired bases can migrate through the run while the DNA polymerase is bound to the template-primer, or the DNA polymerase dissociates from the DNA to allow misalignment but remains tethered to the template through interactions with the clamp. Finally, the T4 replication accessory proteins reduced by >/=10-fold the rate at which exonuclease-deficient T4 DNA polymerase generated deletions of larger numbers of nucleotides, indicating that these proteins influence replication fidelity for other than single base mutations.

Bacteriophage T4↗
PubMed1998

A cell cycle-regulated adenine DNA methyltransferase from Caulobacter crescentus processively methylates GANTC sites on hemimethylated DNA.

The kinetic properties of an adenine DNA methyltransferase involved in cell cycle regulation of Caulobacter crescentus have been elucidated by using defined unmethylated or hemimethylated DNA (DNAHM) substrates. Catalytic efficiency is significantly enhanced with a DNAHM substrate. Biphasic kinetic behavior during methyl incorporation is observed when unmethylated or DNAHM substrates are used, indicating that a step after chemistry limits enzyme turnover and is most likely the release of enzyme from methylated DNA product. The enzyme is thermally inactivated at 30 degrees C within 20 min; this process is substantially decreased in the presence of saturating concentrations of DNAHM, suggesting that the enzyme preferentially binds DNA before S-adenosylmethionine. The activity of the enzyme shows an unusual sensitivity to salt levels, apparently dissociating more rapidly from methylated DNA product as the salt level is decreased. The enzyme acts processively during methylation of specific DNA sequences, indicating a preferred order of product release in which S-adenosylhomocysteine is released from enzyme before fully methylated DNA. The kinetic behavior and activity of the enzyme are consistent with the temporal constraints during the cell cycle-regulated methylation of newly replicated chromosomal DNA.

Caulobacter crescentus↗
PubMed1998

Clamp subunit dissociation dictates bacteriophage T4 DNA polymerase holoenzyme disassembly.

Clamp proteins confer processivity to the DNA polymerase during DNA replication. These oligomeric proteins are loaded onto DNA by clamp loader protein complexes in an ATP-dependent manner. The mechanism by which the trimeric bacteriophage T4 clamp protein (the 45 protein) loads and dissociates from DNA was investigated as a function of its intersubunit protein-protein interactions. These interactions were continuously monitored using a fluorescence resonance energy transfer (FRET) based assay. A cysteine mutant of the 45 protein was constructed to facilitate site-specific incorporation of a fluorescent probe at the subunit interface. This site was chosen such that FRET was observed between the introduced fluorescent probe and a tryptophan residue located on the opposing subunit. By use of this fluorescently labeled 45 protein, it was possible to obtain an estimate of an apparent trimer dissociation constant from either a cooperative (0.08 +/- 0.04 microM2 at 25 degrees C) or a noncooperative (0.51 microM and 0.17 microM at 25 degrees C) model. Upon mixing the fluorescently labeled 45 protein with a 45 protein containing 4-fluorotryptophan, a nonfluorescent tryptophan analogue, subunit exchange between the two variants of the 45 protein was observed according to a reduction in intersubunit FRET. Subunit exchange rate constants measured in the presence or absence of the clamp loader (44/62 complex), the polymerase (43 protein), and/or a primer template DNA substrate demonstrate (a) that the 45 protein is not loaded onto DNA by subunit exchange and (b) that the disassembly dissociation of a stalled holoenzyme from DNA is dictated by 45 protein subunit dissociation.

Bacteriophage T4↗
PubMed1998

Development of an internally quenched fluorescent substrate for Escherichia coli leader peptidase.

Escherichia coli leader peptidase, an integral membrane protein, is responsible for the cleavage of the signal sequence of many exported proteins. Recent studies suggest that it is a novel serine protease that utilizes a serine-lysine catalytic dyad. In an effort to further understand the mechanism of this enzyme, an internally quenched fluorescent peptide substrate incorporating the leader peptidase cleavage site of maltose binding protein signal peptide, Y(NO2)-F-S-A-S-A-L-A-K-I-K(Abz) (anthraniloyl), was designed and synthesized. In the intact peptide, the fluorescence of the anthraniloyl group is quenched by the 3-nitrotyrosine. This quenched fluorescence is liberated upon cleavage of the peptide by the leader peptidase, resulting in increased fluorescence that could then be monitored fluorometrically. The designed substrate can be cleaved effectively by E. coli leader peptidase as detected by both HPLC and fluorescent spectroscopy. Mass spectra of cleavage products demonstrated that the cleavage occurs at the predicted site (A-K). The cleavage of the peptide substrate has a linear dependence on the enzyme concentration (0.1 to 1.9 microM) and the kcat/K(m) was calculated to be 71.1 M-1 s-1. These data are comparable with the unmodified peptide substrate. This report represents the first direct continuous assay based on fluorescence resonance energy transfer for E. coli leader peptidase.

Amino Acid Sequence↗
PubMed1998

Hybrid enzymes: manipulating enzyme design.

Hybrid enzymes are engineered to contain elements of two or more enzymes. Hybrid-enzyme approaches, by taking advantage of the vast array of enzymatic properties that nature has evolved, as well as the strategies that nature has used to evolve them, are becoming an increasingly important avenue for obtaining novel enzymes with desired activities and properties.

Enzymes↗
PubMed1998

Stretching exercises--flexibility in dihydrofolate reductase catalysis.

As an enzyme, dihydrofolate reductase performs two tasks: transformation of its substrate dihydrofolate or folate to tetrahydrofolate, using NADPH as a cofactor, and regeneration of the enzyme for a subsequent round of catalysis. Studies discussed in this review highlight the role of conformational flexibility in both of these enzymatic functions.

Catalysis↗
PubMed1997

Evidence for a functional role of the dynamics of glycine-121 of Escherichia coli dihydrofolate reductase obtained from kinetic analysis of a site-directed mutant.

Two-dimensional heteronuclear (1H-15N) nuclear magnetic relaxation studies of dihydrofolate reductase (DHFR) from Escherichia coli have demonstrated that glycine-121 which is 19 A from the catalytic center of the enzyme has large-amplitude backbone motions on the nanosecond time scale [Epstein, D. M., Benkovic, S. J., and Wright, P. E. (1995) Biochemistry 34, 11037-11048]. In order to probe the dynamic-function relationships of this residue, we constructed a mutant enzyme in which this glycine was changed to valine. Equilibrium binding studies indicated that the Val-121 mutant retained wild-type binding properties with respect to dihydrofolate and tetrahydrofolate; however, binding to NADPH and NADP+ was decreased by 40-fold and 2-fold, respectively, relative to wild-type DHFR. Single-turnover experiments indicated that hydride transfer was reduced by 200-fold to a rate of 1.3 s-1 and was the rate-limiting step in the steady state. Interestingly, pre-steady-state kinetic analysis of the Val-121 mutant revealed a conformational change which preceded chemistry that occurred at a rate of 3.5 s-1. If this step exists in the kinetic mechanism of the wild-type enzyme, then it would be predicted to occur at a rate of approximately 2000 s-1. Glycine-121 was also changed to alanine, serine, leucine, and proline. While the Ala-121 and Ser-121 mutants behaved similar to wild-type DHFR, the Leu-121 and Pro-121 mutants behaved like Val-121 DHFR in that hydride transfer was the rate-limiting step in the steady state and a conformational change preceding chemistry was observed. Finally, insertion of a glycine or valine between amino acids 121 and 122 produced mutant enzymes with properties similar to wild-type or Val-121 DHFRs, respectively. Taken together, these results provide compelling evidence for dynamic coupling of a remote residue to kinetic events at the active site of DHFR.

Binding Sites↗
PubMed1997

Dissociation of bacteriophage T4 DNA polymerase and its processivity clamp after completion of Okazaki fragment synthesis.

The mechanism of bacteriophage T4 DNA polymerase (gp43) and clamp (gp45) protein dissociation from the holoenzyme DNA complex was investigated under conditions simulating the environment encountered upon completion of an Okazaki fragment. Lagging strand DNA synthesis was approximated using a synthetic construct comprised of a doubly biotinylated, streptavidin-bound 62-mer DNA template, paired with complementary primers to generate an internal 12-base gap where the 5'-end primer contained either a 5'-OH (DNA primer) or a 5'-triphosphate (RNA primer) group. Rapid kinetic measurements revealed that upon encountering the blocking primer, the holoenzyme either dissociates from DNA (approximately 40%) or strand-displaces the blocking strand (approximately 60%). The two blocking oligonucleotides (DNA or RNA) induce a 30-50-fold increase in the rate of holoenzyme dissociation, with both polymerase and clamp proteins dissociating simultaneously. Inhibition of ATP hydrolysis by ATP-gamma-S did not have a measurable effect upon holoenzyme dissociation from DNA. The presence of gp32, the single-strand binding protein, caused a small (3-fold) increase in the rate constant for dissociation.

Bacteriophage T4↗
PubMed1997

Consideration of the pH-dependent inhibition of dihydrofolate reductase by methotrexate.

Poisson-Boltzmann calculations were used to determine the pKa of protein functional groups in the unliganded dihydrofolate reductase enzyme, and the pKa of protein and ligand groups in methotrexate-enzyme complexes. The results reported here are in conflict with two fundamental tenets of dihydrofolate reductase inhibition by methotrexate: (1) Asp27 is not expected to be protonated near pH 6.5 in the apoenzyme as previously proposed based on fitting of empirical equations to binding data, and (2) the calculated pKa for the pteridine N1 of the inhibitor while bound to the protein is significantly lower than that estimated for this group from interpretation of NMR data (>10). In fact, the electrostatic calculations and complementary quantum chemical calculations indicate that Asp27 is likely protonated when methotrexate is bound, resulting in a neutral dipole-dipole interaction rather than a salt-bridge between the enzyme and the inhibitor. Reasons for this discrepancy with the experimental data are discussed. Furthermore, His45 and Glu17 in the Escherichia coli enzyme are proposed to be in part responsible for the pH dependence of the conformational degeneracy in the inhibitor-enzyme complex.

4-Aminobenzoic Acid↗
PubMed1997

Mutations in HIV reverse transcriptase which alter RNase H activity and decrease strand transfer efficiency are suppressed by HIV nucleocapsid protein.

Structural studies of authentic HIV reverse transcriptase (RT) suggest a role for the p51 carboxyl terminus in forming an active RNase H conformation [Rodgers, D. W., Gamblin, S. J., Harris, B. A., Ray, S., Culp, J. S., Hellmig, B., Woolf, D. J., Debouck, C. & Harrison, S. C. (1995) Proc. Natl. Acad. Sci. USA 92, 1222-1226]. We have purified mutant RT heterodimers containing deletion of 5, 9, or 13 amino acids from the p51 carboxyl terminus. These "selectively deleted" heterodimers have been analyzed for changes in RNA-dependent DNA polymerase activity, RNase H activity, and the ability to catalyze DNA strand transfer. As deletions extended into the p51 subunit, a decrease in the stability of the RT-DNA complex was apparent. The largest effect was observed for p66/p51Delta13 RT, which showed a 3-fold decrease relative to wild-type RT. RNase H activity was measured by digestion of the RNA in a 5' 32P-labeled RNA/DNA hybrid. Deletion of 5 or 9 amino acids from p51 had little effect on synthesis-dependent and synthesis-independent RNase H activities. In contrast, deletion of 13 amino acids from p51 increased the length of the hydrolysis products of both RNase H activities by 8-10 bp, thus changing the spatial relationship between the polymerase and RNase H active sites from a distance of 17-18 bp to 26-27 bp. The Delta13 derivative was also incapable of efficient DNA strand transfer. This defect in strand transfer could be suppressed by the 71-amino acid form of HIV nucleocapsid protein (NC) but not by the 55-amino acid form (NC55) or by equine infectious anemia virus NC. These results provide evidence for the existence of a specific complex between RT and NC and are discussed in terms of the role of this complex in proviral DNA synthesis.

Base Sequence↗