A stopped flow transient kinetic analysis of substrate binding and catalysis in Escherichia coli D-3-phosphoglycerate dehydrogenase

Pre-steady state, stopped flow analysis of Escherichia coli D-3-phosphoglycerate dehydrogenase was performed by following the fluorescence of protein tryptophan and the fluorescence resonance energy transfer from protein tryptophan to bound NADH. The results indicate that binding of substrates is ordered, with coenzyme, NADH, binding first. Furthermore, the analysis indicated that there are two sets of sites on the tetrameric enzyme that can be differentiated by their kinetic behavior. NADH binding was consistent with an initial binding event followed by a slow conformational change for each site. The slow conformational change is responsible for the apparent tight binding of NADH to the apoenzyme but is too slow to participate in the catalytic cycle when the enzyme is rapidly turning over. Subsequent binding of the substrate, alpha-ketoglutarate, was characterized by a rapid equilibrium binding event followed by a conformational change for each site. Catalysis in the direction of NAD(+) reduction showed a distinct burst of activity followed by a slow rate of turnover, indicating that the rate-limiting step is after hydride transfer. Catalysis in the direction of NADH oxidation did not display burst kinetics, indicating that the rate-limiting step is at or before the hydride transfer step. The burst data indicated that the rate of NAD(+) reduction (3.8 s(-1)) is similar to the k(cat) of the enzyme (2-3 s(-1)) in that direction. However, analysis of the reaction with deuterated NADH failed to show an effect on the velocity of the reaction with a V(H)/V(D)=1.07+/-0.06. None of the other rates determined by stopped flow analysis could account for the k(cat) of the enzyme in either direction (forward k(cat)=0.01 s(-1), reverse k(cat)=2-3 s(-1)), suggesting that the rate-limiting step in both directions is a conformational change in the enzyme that is not detected optically.     

Conformational relaxation following hydride transfer plays a limiting role in dihydrofolate reductase catalysis

The catalytic cycle of an enzyme is frequently associated with conformational changes that may limit maximum catalytic throughput. In Escherichia coli dihydrofolate reductase, release of the tetrahydrofolate (THF) product is the rate-determining step under physiological conditions and is associated with an "occluded" to "closed" conformational change. In this study, we demonstrate that in dihydrofolate reductase the closed to occluded conformational change in the product ternary complex (E.THF.NADP (+)) also gates progression through the catalytic cycle. Using NMR relaxation dispersion, we have measured the temperature and pH dependence of microsecond to millisecond time scale backbone dynamics of the occluded E.THF.NADP (+) complex. Our studies indicate the presence of three independent dynamic regions, associated with the active-site loops, the cofactor binding cleft, and the C-terminus and an adjacent loop, which fluctuate into discrete conformational substates with different kinetic and thermodynamic parameters. The dynamics of the C-terminally associated region is pH-dependent (p K a < 6), but the dynamics of the active-site loops and cofactor binding cleft are pH-independent. The active-site loop dynamics access a closed conformation, and the accompanying closed to occluded rate constant is comparable to the maximum pH-independent hydride transfer rate constant. Together, these results strongly suggest that the closed to occluded conformational transition in the product ternary complex is a prerequisite for progression through the catalytic cycle and that the rate of this process places an effective limit on the maximum rate of the hydride transfer step.     

A kinetic study of the subunit dissociation and reassembly of rabbit muscle phosphofructokinase.

The kinetics of dissociation and reassembly of rabbit skeletal muscle phosphofructokinase has been studied using fluorescence, stopped-flow fluorescence and enzyme activity measurements. The dissociation of the fully active tetramer in 0.8 M guanidine hydrochloride (0.1 M potassium phosphate, pH 8.0) occurs in three kinetic phases as measured by changes in the protein fluorescence emission intensity: dissociation of tetramer to dimer with a relaxation time of a few milliseconds; dissociation of dimer to monomer with a relaxation time of a few seconds; and a conformational change of the monomer with a relaxation time of a few minutes. All three phases exhibit first-order kinetics; ATP (0.05 mM) retards the second step but does not influence the rate of the other two processes. The rate of the second process increases with decreasing temperature; this may be due to the involvement of hydrophobic interactions in the stabilization of the dimeric enzyme. A further unfolding of the monomer polypeptide chain occurs at higher guanidine concentrations, and the relaxation time associated with this process was found to be 83 ms in 2.5 M guanidine, 0.1 M potassium phosphate (pH 8.0) at 23 degrees C. The phosphofructokinase monomers were reassembled from 0.8 M guanidine chloride by 1:10 dilution of the guanidine hydrochloride concentration and yielded a protein with 70-94% of the original activity, depending on the protein concentration. The reactivation process follows second-order kinetics; ATP (5 mM) increases the rate of reactivation without altering the reaction order, while fructose 6-phosphate does not influence the rate of reaction. The rate-determining step is probably the association of monomers to form the dimer.     

Pre-steady state quantification of the allosteric influence of Escherichia coli phosphofructokinase.

Stopped-flow kinetics was utilized to determine how allosteric activators and inhibitors of wild-type Escherichia coli phosphofructokinase influenced the kinetic rate and equilibrium constants of the binding of substrate fructose 6-phosphate. Monitoring pre-steady state fluorescence intensity emission changes upon an addition of a ligand to the enzyme was possible by a unique tryptophan per subunit of the enzyme. Binding of fructose 6-phosphate to the enzyme displayed a two-step process, with a fast complex formation step followed by a relatively slower isomerization step. Systematic addition of fructose 6-phosphate to phosphofructokinase in the absence and presence of several fixed concentrations of phosphoenolpyruvate indicated that the inhibitor binds to the enzyme concurrently with the substrate, forming a ternary complex and inducing a conformational change, rather than a displacement of the equilibrium as predicted by the classical two-state model (Monod, J., Wyman, J., and Changeux, J. P. (1965) J. Mol. Biol. 12, 88-118). The activator, MgADP, also altered the affinity of fructose 6-phosphate to the enzyme by forming a ternary complex. Furthermore, both phosphoenolpyruvate and MgADP act by influencing the fast complex formation step while leaving the slower enzyme isomerization step essentially unchanged.     

Transient state kinetic investigation of 5-aminolevulinate synthase reaction mechanism

5-Aminolevulinate synthase (ALAS), a pyridoxal 5'-phosphate-dependent enzyme, catalyzes the first, and regulatory, step of the heme biosynthetic pathway in nonplant eukaryotes and some bacteria. 5-Aminolevulinate synthase is a dimeric protein having an ordered kinetic mechanism with glycine binding before succinyl-CoA and with aminolevulinate release after CoA and carbon dioxide. Rapid scanning stopped-flow absorption spectrophotometry in conjunction with multiple turnover chemical quenched-flow kinetic analyses and a newly developed CoA detection method were used to examine the ALAS catalytic reaction and identify the rate-determining step. The reaction of glycine with ALAS follows a three-step kinetic process, ascribed to the formation of the Michaelis complex and the pyridoxal 5'-phosphate-glycine aldimine, followed by the abstraction of the glycine pro-R proton from the external aldimine. Significantly, the rate associated with this third step (k(3) = 0.002 s(-1)) is consistent with the rate determined for the ALAS-catalyzed removal of tritium from [2-(3)H(2)]glycine. Succinyl-CoA and acetoacetyl-CoA increased the rate of glycine proton removal approximately 250,000- and 10-fold, respectively, supporting our previous proposal that the physiological substrate, succinyl-CoA, promotes a protein conformational change, which accelerates the conversion of the external aldimine into the initial quinonoid intermediate (Hunter, G. A., and Ferreira, G. C. (1999) J. Biol. Chem. 274, 12222-12228). Rapid scanning stopped-flow and quenched-flow kinetic analyses of the ALAS reaction under single turnover conditions lend evidence for two quinonoid reaction intermediates and a model of the ALAS kinetic mechanism in which product release is at least the partially rate-limiting step. Finally, the carbonyl and carboxylate groups of 5-aminolevulinate play a major protein-interacting role by inducing a conformational change in ALAS and, thus, possibly modulating product release.     

Sensitive fluorogenic substrate for alkaline phosphatase

Alkaline phosphatase serves both as a model enzyme for studies on the mechanism and kinetics of phosphomonoesterases and as a reporter in enzyme-linked immunosorbent assays (ELISAs) and other biochemical methods. The tight binding of the enzyme to its inorganic phosphate product leads to strong inhibition of catalysis and confounds measurements of alkaline phosphatase activity. We have developed an alkaline phosphatase substrate in which the fluorescence of rhodamine is triggered on P–O bond cleavage in a process mediated by a “trimethyl lock.” Although this substrate requires a nonenzymatic second step to manifest fluorescence, we demonstrated that the enzymatic first step limits the rate of fluorogenesis. The substrate enables the catalytic activity of alkaline phosphatase to be measured with high sensitivity and accuracy. Its attributes are ideal for enzymatic assays of alkaline phosphatase for both basic research and biotechnological applications.     

Phosphate dependency of phosphofructokinase 2

Experiments performed at micromolar concentrations of inorganic phosphate support the conclusion that liver phosphofructokinase 2 would be completely inactive in the absence of inorganic phosphate or arsenate. The concentration of inorganic phosphate that allowed half-maximal activity decreased with increasing pH, being approximately 0.11 mM at pH 6.5 and 0.05 mM at pH 8. The effect of phosphate was to increase V and to decrease Km for fructose 6-phosphate, without affecting Km for ATP. Citrate and P-enolpyruvate inhibited the enzyme non-competitively with fructose 6-phosphate and independently of the concentration of inorganic phosphate. Phosphorylation of the enzyme by the catalytic subunit of cyclic-AMP-dependent protein kinase did not markedly modify the phosphate requirement and its effect of inactivating phosphofructokinase 2 could not be counteracted by excess phosphate. A nearly complete phosphate dependency was also observed with phosphofructokinase 2 purified from Saccharomyces cerevisiae or from spinach leaves. By contrast, the fructose 2,6-bisphosphatase activity of the liver bifunctional enzyme was not dependent on the presence of inorganic phosphate. Phosphate increased this activity about threefold when measured in the absence of added fructose 6-phosphate and a half-maximal effect was reached at approximately 0.5 mM phosphate. Like glycerol phosphate, phosphate counteracted the inhibition of fructose 2,6-bisphosphatase by fructose 6-phosphate, but a much higher concentration of phosphate than of glycerol phosphate was required to reach this effect.     

Detection of conformational changes along the kinetic pathway of protein kinase A using a catalytic trapping technique.

The dissociation rate constants for the two products of the reaction catalyzed by protein kinase A, ADP and phosphopeptide, were measured using a catalytic trapping technique to determine the role of product release in enzyme turnover. The enzyme was preequilibrated with ADP, and the reaction was initiated with a peptide substrate, LRRASLG, and ATP in a rapid quench flow instrument. At high, free magnesium concentrations (>2 mM), the large 'burst' in phosphopeptide production disappears, and, at low concentrations of free magnesium (0.5-1 mM), the kinetic transients become sigmoidal prior to the linear turnover phase. Increasing the concentrations of ATP or ADP did not influence the shape of the kinetic transients in the first 20 ms. ADP preequilibration protects the enzyme from inhibition by the covalent inactivator p-fluorosulfonylbenzoyl 5'-adenosine at 0.5 mM free magnesium, indicating that a competent E. ADP complex forms at low metal concentrations and the sigmoidal behavior in the catalytic trapping experiment is not due to free enzyme at high ATP concentrations. Simulations of the data indicate that ADP release is rate-limiting for turnover at high magnesium concentrations, but, at lower physiological levels of 0.5 and 1 mM, the off rate of ADP is 3- and 2-fold higher than k(cat), respectively. In contrast, the initial portions of the kinetic transients at 0.5 mM free magnesium were unaffected by phosphopeptide preequilibration, indicating that the release rate of this product is significantly larger than turnover. The transient kinetic data, coupled with a previous report [Shaffer and Adams (1999) Biochemistry 38, 5572-5581], support a phosphorylation mechanism under physiological magnesium concentrations that incorporates two partially rate-determining conformational changes, one prior to and one after the phosphoryl transfer step. We propose that the initial step activates the enzyme through key positioning of one or more active-site residues and the second step relaxes this conformation, a prerequisite for a subsequent catalytic cycle.     

Snapshots of catalysis: the structure of fructose-1,6-(bis)phosphate aldolase covalently bound to the substrate dihydroxyacetone phosphate.

Fructose-1,6-bis(phosphate) aldolase is an essential glycolytic enzyme found in all vertebrates and higher plants that catalyzes the cleavage of fructose 1,6-bis(phosphate) (Fru-1,6-P(2)) to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (DHAP). Mutations in the aldolase genes in humans cause hemolytic anemia and hereditary fructose intolerance. The structure of the aldolase-DHAP Schiff base has been determined by X-ray crystallography to 2.6 A resolution (R(cryst) = 0.213, R(free) = 0.249) by trapping the catalytic intermediate with NaBH(4) in the presence of Fru-1,6-P(2). This is the first structure of a trapped covalent intermediate for this essential glycolytic enzyme. The structure allows the elucidation of a comprehensive catalytic mechanism and identification of a conserved chemical motif in Schiff-base aldolases. The position of the bound DHAP relative to Asp33 is consistent with a role for Asp33 in deprotonation of the C4-hydroxyl leading to C-C bond cleavage. The methyl side chain of Ala31 is positioned directly opposite the C3-hydroxyl, sterically favoring the S-configuration of the substrate at this carbon. The "trigger" residue Arg303, which binds the substrate C6-phosphate group, is a ligand to the phosphate group of DHAP. The observed movement of the ligand between substrate and product phosphates may provide a structural link between the substrate cleavage and the conformational change in the C-terminus associated with product release. The position of Glu187 in relation to the DHAP Schiff base is consistent with a role for the residue in protonation of the hydroxyl group of the carbinolamine in the dehydration step, catalyzing Schiff-base formation. The overlay of the aldolase-DHAP structure with that of the covalent enzyme-dihydroxyacetone structure of the mechanistically similar transaldolase and KDPG aldolase allows the identification of a conserved Lys-Glu dyad involved in Schiff-base formation and breakdown. The overlay highlights the fact that Lys146 in aldolase is replaced in transaldolase with Asn35. The substitution in transaldolase stabilizes the enamine intermediate required for the attack of the second aldose substrate, changing the chemistry from aldolase to transaldolase.     

Engineering E. coli Alkaline Phosphatase Yields Changes of Catalytic Activity,Thermal Stability and Phosphate Inhibition

To investigate the function of aspartic acid residue 101 and arginine residue 166 in the active site of Escherichia coli alkaline phosphatase (EAP), two single mutants D101S (Asp 101 &#77 Ser) and R166K (Arg 166 &#77 Lys) and a double mutant D101S/R166K of EAP were generated through site-directed mutagenesis based on over-lap PCR method. Their enzymatic kinetic properties, thermal stabilities and possible reaction mechanism were explored. In the presence of inorganic phosphate acceptor, 1 M diethanolamine buffer, the k cat for D101S mutant enzyme increased 10-fold compared to that of wild-type EAP. The mutant R166K has a 2-fold decrease of k cat relative to the wild-type EAP, but the double mutant D101S/R166K was in the middle of them, indicative of an additive effect of these two mutations. On the other hand, the catalytic efficiencies of mutant enzymes are all reduced because of a substantial increase of K m values. All three mutants were more resistant to phosphate inhibitor than the wild-type enzyme. The analysis of the kinetic data suggests that (1) the D101S mutant enzyme obtains a higher catalytic activity by allowing a faster release of the product; (2) the R166K mutant enzyme can reduce the binding of the substrate and phosphate competitive inhibitor; (3) the double mutant enzyme has characteristics of both quicker catalytic turnover number and decreased affinity for competitive inhibitor. Additionally, pre-steady-state kinetics of D101S and D101S/R166K mutants revealed a transient burst followed by a linear steady state phase, obviously different from that of wild-type EAP, suggesting that the rate-limiting step has partially change from the release of phosphate from non-covalent E-Pi complex to the hydrolysis of covalent E-Pi complex for these two mutants.     

Engineering E. coli Alkaline Phosphatase Yields Changes of Catalytic Activity, Thermal Stability and Phosphate Inhibition

To investigate the function of aspartic acid residue 101 and arginine residue 166 in the active site of Escherichia coli alkaline phosphatase (EAP), two single mutants D101S (Asp 101 →Ser) and R166K (Arg 166 →Lys) and a double mutant D101S/R166K of EAP were generated through site-directed mutagenesis based on over-lap PCR method. Their enzymatic kinetic properties, thermal stabilities and possible reaction mechanism were explored. In the presence of inorganic phosphate acceptor, 1 M diethanolamine buffer, the k cat for D101S mutant enzyme increased 10-fold compared to that of wild-type EAP. The mutant R166K has a 2-fold decrease of k cat relative to the wild-type EAP, but the double mutant D101S/R166K was in the middle of them, indicative of an additive effect of these two mutations. On the other hand, the catalytic efficiencies of mutant enzymes are all reduced because of a substantial increase of K m values. All three mutants were more resistant to phosphate inhibitor than the wild-type enzyme. The analysis of the kinetic data suggests that (1) the D101S mutant enzyme obtains a higher catalytic activity by allowing a faster release of the product; (2) the R166K mutant enzyme can reduce the binding of the substrate and phosphate competitive inhibitor; (3) the double mutant enzyme has characteristics of both quicker catalytic turnover number and decreased affinity for competitive inhibitor. Additionally, pre-steady-state kinetics of D101S and D101S/R166K mutants revealed a transient burst followed by a linear steady state phase, obviously different from that of wild-type EAP, suggesting that the rate-limiting step has partially change from the release of phosphate from non-covalent E-Pi complex to the hydrolysis of covalent E-Pi complex for these two mutants.     

SRC tail phosphorylation is limited by structural changes in the regulatory tyrosine kinase Csk

Src family tyrosine kinases are down-regulated through phosphorylation of a single C-terminal tyrosine by the nonreceptor tyrosine kinase Csk. Despite the fundamental role of Csk in controlling cell growth and differentiation, it is unclear what limits this key signaling reaction and controls the production of catalytically repressed Src. To investigate this issue, stopped-flow fluorescence experiments were performed to determine which steps modulate catalysis. Both Src binding and phosphorylation can be monitored by changes in intrinsic tryptophan fluorescence. Association kinetics are biphasic with the initial phase corresponding to the bimolecular interaction of both proteins and the second phase representing a slow conformational change that coincides with the rate of maximum turnover. The kinetic transients for the phosphorylation reaction are also biphasic with the initial phase corresponding to the rapid phosphorylation and the release of phospho-Src. These data, along with equilibrium sedimentation and product inhibition experiments, suggest that steps involving Src association, phosphorylation, and product release are fast and that a structural change in Csk participates in limiting the catalytic cycle.     

Proteasome accessory factor A (PafA) transferase activity makes sense in the light of its homology with glutamine synthetase

The Pup-proteasome system (PPS) is a prokaryotic tagging and degradation system analogous in function to the ubiquitin-proteasome system (UPS). Like ubiquitin, Pup is conjugated to proteins, tagging them for proteasomal degradation. However, in the PPS, a single Pup-ligase, PafA, conjugates Pup to a wide variety of proteins. PafA couples ATP hydrolysis to formation of an isopeptide bond between Pup and a protein lysine via a mechanism similar to that used by glutamine synthetase (GS) to generate glutamine from ammonia and glutamate. GS can also transfer the glutamyl moiety from glutamine to a hydroxyl amine in an ATP-independent manner. Recently, the ability of PafA to transfer Pup from one protein to another was demonstrated. Here, we report that such PafA activity mechanistically resembles the transferase activity of GS. Both PafA and GS transferase activities are ATP-independent and proceed in two catalytic steps. In the first step catalyzed by PafA, an inorganic phosphate is used by the enzyme to depupylate a Pup donor, while forming an acyl phosphate Pup intermediate. The second step consists of Pup conjugation to the new protein, alongside the release of an inorganic phosphate. Detailed experimental analysis, combined with kinetic modeling of PafA transferase activity, allowed us to correctly predict the kinetics and magnitude of Pup transfer between two targets, and analyze the effects of their affinity to PafA on the efficiency of transfer. By deciphering the mechanism of the PafA transferase reaction in kinetic detail, this work provides in-depth mechanistic understanding of PafA, a key PPS enzyme.     

Characterization of the DNA- and dNTP-binding activities of the human cytomegalovirus DNA polymerase catalytic subunit UL54

The catalytic subunit of the human cytomegalovirus DNA polymerase is critical for the replication of the virus. In the present study, we report the expression and purification of a recombinant catalytic subunit of the human cytomegalovirus DNA polymerase expressed in bacteria which retains polymerase activity. As a first step towards elucidating the nature of the interaction between the enzyme, DNA and dNTPs, we have utilized endogenous tryptophan fluorescence to evaluate the binding of ligands to the enzyme. Using this technique, we demonstrate that the minimal DNA-binding site of the enzyme is 6 nt. We also report the first detailed study of the binding kinetics and thermodynamic parameters involved in the interaction between the enzyme, DNA and dNTPs. Our thermodynamic analyses indicate that the initial formation of the enzyme-DNA binary complex is driven by a favourable entropy change, but is also clearly associated with an unfavourable enthalpic contribution. In contrast, the interaction of dNTPs to the binary complex was shown to depend on a completely different mode of binding that is dominated by a favourable enthalpy change and associated with an unfavourable entropy change. In order to provide additional insights into the structural modifications that occur during catalysis, we correlated the effect of DNA and dNTP binding on protein structure using CD. Our results indicate that the enzyme undergoes a first conformational change upon the formation of the protein-DNA binary complex, which is followed by a second structural modification upon dNTP binding. The present study provides a better understanding of the molecular basis of DNA and dNTP recognition by the catalytic subunit of the human cytomegalovirus DNA polymerase.     

Kinetics of inactivation of green crab (Scylla Serrata) alkaline phosphatase during removal of zinc ions by ethylenediaminetetraacetic acid disodium

Green crab (Scylla Serrata) alkaline phosphatase (EC 3.1.3.1.) is a metalloenzyme, the each active site in which contains a tight cluster of two zinc ions and one magnesium ion. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity previously described by Tsou has been applied to a study on the kinetics of the course of inactivation of the enzyme by ethylenediaminetetraacetic acid disodium (EDTA). The kinetics of the substrate reaction with different concentrations of the substrate p-nitrophenyl phosphate (PNPP) and inactivator EDTA suggested a complexing mechanism for inactivation by, and substrate competition with, EDTA at the active site. The inactivation kinetics are single phasic, showing the initial formation of an enzyme-EDTA complex is a relatively rapid reaction, followed a slow inactivation step that probably involves a conformational change of the enzyme. Zinc ions are finally removed from the enzyme. The presence of metal ions apparently stabilizes an active-site conformation required for enzyme activity.     

Dimer asymmetry and the catalytic cycle of alkaline phosphatase from Escherichia coli.

Although alkaline phosphatase (APase) from Escherichia coli crystallizes as a symmetric dimer, it displays deviations from Michaelis-Menten kinetics, supported by a model describing a dimeric enzyme with unequal subunits [Orhanovi? S., Pavela-Vrancic M. and Flogel-Mrsi? M. (1994) Acta. Pharm.44, 87-95]. The possibility, that the observed asymmetry could be attributed to negative cooperativity in Mg2+ binding, has been examined. The influence of the metal ion content on the catalytic properties of APase from E. coli has been examined by kinetic analyses. An activation study has indicated that Mg2+ enhances APase activity by a mechanism that involves interactions between subunits. The observed deviations from Michaelis-Menten kinetics are independent of saturation with Zn2+ or Mg2+ ions, suggesting that asymmetry is an intrinsic property of the dimeric enzyme. In accordance with the experimental data, a model describing the mechanism of substrate hydrolysis by APase has been proposed. The release of the product is enhanced by a conformational change generating a subunit with lower affinity for both the substrate and the product. In the course of the catalytic cycle the conformation of the subunits alternates between two states in order to enable substrate binding and product release. APase displays higher activity in the presence of Mg2+, as binding of Mg2+ increases the rate of conformational change. A conformationally controlled and Mg2+-assisted dissociation of the reaction product (Pi) could serve as a kinetic switch preventing loss of Pi into the environment.     

Conformational selection and induced changes along the catalytic cycle of Escherichia coli dihydrofolate reductase

Protein function often involves changes between different conformations. Central questions are how these conformational changes are coupled to the binding or catalytic processes during which they occur, and how they affect the catalytic rates of enzymes. An important model system is the enzyme dihydrofolate reductase (DHFR) from Escherichia coli, which exhibits characteristic conformational changes of the active‐site loop during the catalytic step and during unbinding of the product. In this article, we present a general kinetic framework that can be used (1) to identify the ordering of events in the coupling of conformational changes, binding, and catalysis and (2) to determine the rates of the substeps of coupled processes from a combined analysis of nuclear magnetic resonance R₂ relaxation dispersion experiments and traditional enzyme kinetics measurements. We apply this framework to E. coli DHFR and find that the conformational change during product unbinding follows a conformational‐selection mechanism, that is, the conformational change occurs predominantly prior to unbinding. The conformational change during the catalytic step, in contrast, is an induced change, that is, the change occurs after the chemical reaction. We propose that the reason for these conformational changes, which are absent in human and other vertebrate DHFRs, is robustness of the catalytic rate against large pH variations and changes to substrate/product concentrations in E. coli. Proteins 2012;. © 2012 Wiley Periodicals, Inc.     

A pre-steady-state kinetic analysis of substrate binding to human recombinant deoxycytidine kinase: a model for nucleoside kinase action.

Deoxycytidine kinase (dCK) is an enzyme with broad substrate specificity which can phosphorylate pyrimidine and purine deoxynucleosides, including important antiviral and cytostatic agents. In this study, stopped-flow experiments were used to monitor intrinsic fluorescence changes induced upon binding of various phosphate donors (ATP, UTP, and the nonhydrolyzable analogue AMP-PNP) and the acceptor dCyd to recombinant dCK. Monophasic kinetics were observed throughout. The nucleotides as well as dCyd bound to the enzyme by a two-step mechanism, involving a rapid initial equilibrium step, followed by a protein conformational change that is responsible for the fluorescence change. The bimolecular association rate constants for nucleotide binding [(4-10) x 10(3) M-1 s-1] were 2-3 orders of magnitude lower than those for dCyd binding [(1.3-1.5 x 10(6) M-1 s-1]. This difference most likely is due predominantly to the large difference in the forward rate constants of the conformational changes (0.04-0.26 s-1 vs 560-710 s-1). Whereas the kinetics of the binding of ATP, UTP, and AMP-PNP to dCK showed some differences, UTP exhibiting the tightest binding, no significant differences were observed for the binding of dCyd to dCK in the presence or absence of phosphate donors. However, the binding of dCyd to dCK in the presence of ATP or UTP was accompanied by a 1.5- or 3-fold higher quenching amplitude as compared with dCyd alone or in the presence of AMP-PNP. We conclude that ATP and UTP induce a conformational change in the enzyme, thereby enabling efficient phosphoryl transfer.     

Unzipping mechanism of the double-stranded DNA unwinding by a hexameric helicase: quantitative analysis of the rate of the dsDNA unwinding, processivity and kinetic step-size of the Escherichia coli DnaB helicase using rapid quench-flow method

Kinetics of the double-stranded (ds) DNA unwinding by the Escherichia coli replicative helicase DnaB protein has been examined under single-turnover conditions using the chemical quench-flow technique. The unwinding reaction proceeds through an initial conformational transition followed by the unwinding catalytic steps and the release of the single-stranded (ss) DNA. Analyses of the reaction as a function of the number of base-pairs in the dsDNA reveal that the number of catalytic steps is not strictly proportional to the length of the dsDNA. As the helicase approaches the end of the substrate, the remaining approximately 11 bp of the DNA melts without catalytic participation of the enzyme. The kinetic step-size of the DnaB helicase, i.e. the number of the base-pairs unwound in a single catalytic step is only 1.4(+/- 0.2). The low value of the step-size indicates that the helicase unwinds a single base-pair in a single catalytic step. Thus, the DnaB helicase unzips the dsDNA in a reverse process to the zipping mechanism of the non-enzymatic double helix formation. The protein is a fast helicase that at 25 degrees C unwinds approximately 291 bp/s, much faster than previously thought, and the unwinding rate can be much higher at higher temperatures. However, the ATP-state of the enzyme has an increased dissociation rate, resulting in only a moderate unwinding processivity, P = 0.89(+/- 0.03), little dependent on the temperature. The conformational transition of the DnaB helicase-DNA complex, preceding the unwinding, is an intrinsic transition of the enzyme from the stationary conformation to the ATP-state of the helicase.     

Chloroplast Phosphofructokinase: II. Partial Purification, Kinetic and Regulatory Properties

Chloroplast phosphofructokinase from spinach (Spinacia oleracea L.) was purified approximately 40-fold by a combination of fractionations with ammonium sulfate and acetone followed by chromatography on DEAE-Sephadex A-50. Positive cooperative kinetics was observed for the interaction between the enzyme and the substrate fructose 6-phosphate. The optimum pH shifted from 7.7 toward 7.0 as the fructose 6-phosphate concentration was taken below 0.5 mm. The second substrate was MgATP(2-) (Michaelis constant 30 mum). Free ATP inhibited the enzyme. Chloroplast phosphofructokinase was sensitive to inhibition by low concentration of phosphoenolpyruvate and glycolate 2-phosphate (especially at higher pH); these compounds inhibited in a positively cooperative fashion. Inhibitions by glycerate 2-phosphate (and probably glycerate 3-phosphate), citrate, and inorganic phosphate were also recorded; however, inorganic phosphate effectively relieved the inhibitions by phosphoenolpyruvate and glycolate 2-phosphate. These regulatory properties are considered to complement those of ADP-glucose pyrophosphorylase and fructosebisphosphatase in the regulation of chloroplast starch metabolism.