Kinetic analyses of mutations in the glycine-rich loop of cAMP-dependent protein kinase

The conserved glycines in the glycine-rich loop (Leu-Gly50-Thr-Gly52-Ser-Phe-Gly55-Arg-Val) of the catalytic (C) subunit of cAMP-dependent protein kinase were each mutated to Ser (G50S, G52S, and G55S). The effects of these mutations were assessed here using both steady-state and pre-steady-state kinetic methods. While G50S and G52S reduced the apparent affinity for ATP by approximately 10-fold, substitution at Gly55 had no effect on nucleotide binding. In contrast to ATP, only mutation at position 50 interfered with ADP binding. These three mutations lowered the rate of phosphoryl transfer by 7-300-fold. The combined data indicate that G50 and G52 are the most critical residues in the loop for catalysis, with replacement at position 52 being the most extreme owing to a larger decrease in the rate of phosphoryl transfer (29 vs 1.6 s-1 in contrast to 500 s-1 for wild-type C). Surprisingly, all three mutations lowered the affinity for Kemptide by approximately 10-fold, although none of the loop glycines makes direct contact with the substrate. The inability to correlate the rate constant for net product release with the dissociation constant for ADP implies that other steps may limit the decomposition of the ternary product complex. The observations that G52S (a) selectively affects ATP binding and (b) significantly lowers the rate of phosphoryl transfer without making direct contact with either the nucleotide or the peptide imply that this residue serves a structural role in the loop, most likely by positioning the backbone amide of Ser53 for contacting the gamma-phosphate of ATP. Energy-minimized models of the mutant proteins are consistent with the observed kinetic consequences of each mutation. The models predict that only mutation of Gly52 will interfere with the observed hydrogen bonding between the backbone and ATP.     

Release of isolated single kinesin molecules from microtubules

Previous studies on the motor enzyme kinesin suggesting that the enzyme molecule tightly binds to a microtubule by only one of its two mechanochemical head domains were performed with multiple kinesin molecules on each microtubule, raising the possibility that interactions between adjacent bound molecules may interfere with the binding of the second head. To characterize the microtubule-bound state of isolated single kinesin molecules, we have measured the rates of nucleotide-induced dissociation of the complex between microtubules and bead-labeled single molecules of the dimeric kinesin derivative K448-BIO using novel single-molecule kinetic methods. Complex dissociation by <2 microM ADP displays an apparent second-order rate constant of 1.2 x 10(4) M-1 s-1. The data suggest that only one of the two heads is bound to the microtubule in the absence of ATP, that binding of a single ADP to the complex is sufficient to induce dissociation, and that even lengthy exposure of kinesin to the microtubule fails to produce significant amounts of a two-head-bound state under the conditions used. The inhibitor adenylyl imidodiphosphate (AMP-PNP) induces stochastic pauses in the movement of bead-labeled enzyme molecules in 1 mM ATP. Exit from pauses occurs at 2 s-1 independent of AMP-PNP concentration. The same rate constant is obtained for dissociation of the transient kinesin-microtubule complexes formed in 1 mM ADP, 0.5 mM AMP-PNP, suggesting that release of a single AMP-PNP molecule from the enzyme is the common rate-limiting step of the two processes. The results are consistent with alternating-sites movement mechanisms in which two-head-bound states do not occur in the enzyme catalytic cycle until after ATP binding.     

Kinetic mechanism of monomeric non-claret disjunctional protein (Ncd) ATPase

The non-claret disjunctional protein (Ncd) is a kinesin-related microtubule motor that moves toward the negative end of microtubules. The kinetic mechanism of the monomer motor domain, residues 335-700, satisfied a simple scheme for the binding of 2'-3'-O-(N-methylanthraniloyl) (MANT) ATP, the hydrolysis step, and the binding and release of MANT ADP, where T, D, and Pi refer to nucleotide triphosphate, nucleotide diphosphate, and inorganic phosphate, respectively, and MtN is the complex of an Ncd motor domain with a microtubule site. Rate constants k1 and k-4 are the rates of a first order step, an isomerization induced by nucleotide binding. The apparent second order rate constants for the binding steps are 1.5 x 10(6) M-1 s-1 for MANT ATP and 3.5 x 10(6) M-1 s-1 for MANT ADP (conditions, 50 mM NaCl, pH 6.9, 21 degrees C). The rate constant of the hydrolysis step (k2) was obtained from quench flow measurements of the phosphate burst phase corrected for the contribution of the rate of product release to the transient rate constant. The rate of phosphate dissociation was not measured; the value was assigned to account for a steady state rate of 3 s-1. The MtN complex is dissociated by ATP at a rate of 10 s-1 based on light scattering measurements. Dissociation constants of Ncd-nucleotide complexes from microtubules increased in the order adenosine 5'-O-(thiotriphosphate) (ATPgammaS) < ADP-AlF4 < ATP < ADP < ADP-vanadate. Comparison of the properties of Ncd with a monomeric kinesin K332 (Ma and Taylor (1997) J. Biol. Chem. 272, 717-723) showed a close similarity, except that the rate constants for the hydrolysis and ADP release steps and the steady state rate are approximately 15-20 times smaller for Ncd. There are two differences that may affect the reaction pathway. The rate of dissociation of MtN by ATP is comparable to the rate of the hydrolysis step, and N.T may dissociate in the cycle, whereas for kinesin, dissociation occurs after hydrolysis. The rate of dissociation of MtN by ADP is larger than the rate of ADP release from MtN.D, whereas for the microtubule-kinesin complex, the rate of dissociation by ADP is smaller than the rate of ADP release. The monomeric Mt.Ncd complex is not processive.     

Stopped-flow kinetic investigations of conformational changes of pig kidney Na+,K+-ATPase

The kinetics of Na+-dependent partial reactions of the Na+,K+-ATPase were investigated via the stopped-flow technique using the fluorescent labels RH421 and BIPM. After the enzyme is mixed with MgATP, both labels give almost identical kinetic responses. Under the chosen experimental conditions two exponential time functions are necessary to fit the data. The dominant fast phase, 1/tau1 approximately 180 s-1 (saturating [ATP] and [Na+], pH 7.4 and 24 degrees C), is attributed to phosphorylation of the enzyme and a subsequent conformational change (E1ATP(Na+)3 --> E2P(Na+)3 + ADP). The rate of the phosphorylation reaction measured by the acid quenched-flow technique was 190 s-1 at 100 microM ATP, suggesting that phosphorylation controls the kinetics of the RH421 signal and that the conformational change is very fast (>/=600 s-1). The rate of the RH421 signal was optimal at pH 7.5. The Na+ concentration dependence of 1/tau1 showed half-saturation at a Na+ concentration of 8-10 mM with positive cooperativity involved in the occupation of the Na+ binding sites. The apparent dissociation constant of the high affinity ATP binding site determined from the ATP concentration dependence of 1/tau1 was 7.0 (+/-0.6) microM, while the apparent Kd for the low affinity site and the rate constant for the E2 to E1 conformational change evaluated in the absence of Mg2+ were 143 (+/-17) microM and 相似文献   

Kinetic competence of a phosphoryl enzyme intermediate in the glucose-1,6-p2 synthase-catalyzed reaction. Purification, properties, and kinetic studies

Glucose-1,6-P2 synthase of beef brain which catalyzes the formation of glucose-1,6-P2 and glycerate-3-P from glycerate-1,3-P2 and glucose-1-P has been purified 700-fold with an overall recovery of 19%. The purification procedure involves an ammonium sulfate fractionation of the crude extract, DE52 and hydroxylapatite column chromatography and isoelectric focusing. The isolated enzyme appears to be homogeneous by sodium dodecyl sulfate gel electrophoresis. Its molecular weight is estimated to be about 70,000 by gel filtration on Sephadex G-200 which agrees with the value obtained by sodium dodecyl sulfate gel electrophoresis. A phosphoryl enzyme intermediate in the catalytic reaction is indicated by the following evidence: glycerate-1,3-P2[1-32P] labels the enzyme. The label is removed by acceptor substrates such as glucose-1-P. Using a rapid quenching device at 23 degrees and pH 8.0, the first order rate constant for phosphorylation of the enzyme is 20 s-1, compared with an overall rate with the best acceptor, glucose-1-P, of 19 s-1. Dephosphorylation by glucose-1-P is at 37 s-1. Mg2+ is required for both phosphoryl transfers and the overall reaction. In the complete reaction the fraction of enzyme that is phosphorylated depends on the concentrations of glycerate-1,3-P2 and the concentration and nature of the acceptor in a way that could be predicted from the steady state parameters, the Km values, and the kinetic constants observed for the single turnover. Reciprocal plots of initial rates as a function of both substrate concentrations are families of parallel lines. The 32P-labeled phosphoryl enzyme intermediate was found to be acid-stable and somewhat alkaline-labile. Phosphoserine was identified from the partial acid hydrolysate of a protease digest of [32P] phosphoryl enzyme by two-dimensional thin layer chromatography.     

The Escherichia coli FOF1 gammaM23K uncoupling mutant has a higher K0.5 for Pi. Transition state analysis of this mutant and others reveals that synthesis and hydrolysis utilize the same kinetic pathway

The Escherichia coli FOF1 ATP synthase uncoupling mutation, gammaM23K, was found to increase the energy of interaction between gamma and beta subunits, prevent the proper utilization of binding energy to drive catalysis, and block the enzyme in a Pi release mode. In this paper, the effects of this mutation on substrate binding in cooperative ATP synthesis are assessed. Activation of ATP synthesis by ADP and Pi was determined for the gammaM23K FOF1. The K0.5 for ADP was not affected, but K0.5 for Pi was approximately 7-fold higher even though the apparent Vmax was close to the wild-type level. Wild-type enzyme had a turnover number of 82 s-1 at pH 7.5 and 30 degrees C. During oxidative phosphorylation, the apparent dissociation constant (KI) for ATP was not affected and was 5-6 mM for both wild-type and gammaM23K enzymes. Thus, the apparent binding affinity for ATP in the presence of DeltamuH+ was lowered by 7 orders of magnitude from the affinity measured at the high-affinity catalytic site. Arrhenius analysis of ATP synthesis for the gammaM23K FOF1 revealed that, like those of ATP hydrolysis, the transition state DeltaH was much more positive and TDeltaS was much less negative, adding up to little change in DeltaG. These results suggested that ATP synthesis is inefficient because of an extra bond between gamma and beta subunits which must be broken to achieve the transition state. Analysis of the transition state structures using isokinetic plots demonstrate that ATP hydrolysis and synthesis utilize the same kinetic pathway. Incorporating this information into a model for rotational catalysis suggests that at saturating substrate concentrations, the rate-limiting step for hydrolysis and synthesis is the rotational power stroke where each of the beta subunits changes conformation and affinity for nucleotide.     

A kinetic analysis of the oligonucleotide-modulated ATPase activity of the helicase domain of the NS3 protein from hepatitis C virus. The first cycle of interaction of ATP with the enzyme is unique

Hepatitis C virus (HCV) helicase (E) formed spectrofluorometrically detectable complexes with a 16-mer and HF16 (a 16-mer with 5'-hexachlorofluoresceinyl moiety). The interaction of helicase with these effectors was investigated by kinetic techniques to determine if the complexes were kinetically competent for ATP hydrolysis. kcat values with the 16-mer and HF16 were 2.7 and 36 s-1, respectively. The maximal value of the rate constant for the approach of an intermediate to the steady-state level has to be at least 4-fold greater than kcat for it to be kinetically competent. This value was 1.2 s-1 with HF16 and "E.ATP" and was 1.82 s-1 with ATP and E.HF16. These values were too small for formation of these intermediates to be kinetically competent in ATP hydrolysis. Dissociation of "E.HF16. ATP" (0.34 s-1) was also too slow to contribute significantly to catalysis. Furthermore, the Km of E.HF16 for ATP (3 mircoM) was significantly less than the Km for ATP hydrolysis at a saturating concentration of HF16 (320 microM). HCV helicase has two nucleotide-binding sites per monomer. If the fluorescence changes observed were associated with structure changes preceding steady-state catalysis (isomerization), pre-steady-state data could be reconciled with the turnover data. Data for the 16-mer yielded similar conclusions.     

Kinetics of Na(+)-dependent conformational changes of rabbit kidney Na+,K(+)-ATPase

The kinetics of Na(+)-dependent partial reactions of the Na+,K(+)-ATPase from rabbit kidney were investigated via the stopped-flow technique, using the fluorescent labels N-(4-sulfobutyl)-4-(4-(p-(dipentylamino)phenyl)butadienyl)py ridinium inner salt (RH421) and 5-iodoacetamidofluorescein (5-IAF). When covalently labeled 5-IAF enzyme is mixed with ATP, the two labels give almost identical kinetic responses. Under the chosen experimental conditions two exponential time functions are necessary to fit the data. The dominant fast phase, 1/tau 1 approximately 155 s-1 for 5-IAF-labeled enzyme and 1/tau 1 approximately 200 s-1 for native enzyme (saturating [ATP] and [Na+], pH 7.4 and 24 degrees C), is attributed to phosphorylation of the enzyme and a subsequent conformational change (E1ATP(Na+)3-->E2P(Na+)3 + ADP). The smaller amplitude slow phase, 1/tau 2 = 30-45 s-1, is attributed to the relaxation of the dephosphorylation/rephosphorylation equilibrium in the absence of K+ ions (E2P<==>E2). The Na+ concentration dependence of 1/tau 1 showed half-saturation at a Na+ concentration of 6-8 mM, with positive cooperatively involved in the occupation of the Na+ binding sites. The apparent dissociation constant of the high-affinity ATP-binding site determined from the ATP concentration dependence of 1/tau 1 was 8.0 (+/- 0.7) microM. It was found that P3-1-(2-nitrophenyl)ethyl ATP, tripropylammonium salt (NPE-caged ATP), at concentrations in the hundreds of micromolar range, significantly decreases the value of 1/tau 1, observed. This, as well as the biexponential nature of the kinetic traces, can account for previously reported discrepancies in the rates of the reactions investigated.     

A second magnesium ion is critical for ATP binding in the kinase domain of the oncoprotein v-Fps

The activity of the kinase domain of the oncoprotein v-Fps was found to be sensitive to the concentration of magnesium ions. Plots of initial velocity versus free magnesium concentration are hyperbolic and do not extrapolate to the origin at stoichiometric ATP-Mg, indicating that there are two sites for metal chelation on the enzyme and the second is nonessential for catalysis. The second metal is strongly activating and increases the reaction rate constant almost 20-fold from 0.5 to 8.3 s-1 using 0.2 mM ATP-Mg and 1 mM peptide, EAEIYEAIE. This increase in rate is due to a large increase in the apparent affinity of ATP-Mg at high magnesium concentrations. At 0.5 and 10 mM free Mg2+, KATP-Mg is 3.6 and 0.22 mM, respectively. Extrapolation of the observed affinity of ATP-Mg to zero and infinite free metal indicates that KATP-Mg is greater than 8 mM in the absence of the second metal and 0.1 mM in the presence of the second metal, a minimum 80-fold enhancement. By comparison, free levels of the divalent ion do not influence maximum turnover (kcat) and have only a 2-fold effect on the Km for the peptide substrate between 0.5 and 20 mM free Mg2+. Viscosometric studies indicate that free Mg2+ does not influence the rates of phosphoryl transfer or net product release above 0.5 mM but does affect directly the dissociation constant for ATP-Mg. The Kd for ATP-Mg in the absence and presence of the second metal ion is >32 and 0.4 mM, respectively. At high magnesium concentrations, ATP-Mg and the peptide substrate bind independently, while at lower concentrations (0.5 mM), there is significant negative binding synergism suggesting that the second metal may help to reduce charge repulsion between ATP-Mg and the peptide. The data indicate that the first metal is sufficient for phosphoryl transfer. While the second metal could have some influence on phosphoryl transfer or product binding, it is a potent activator that functions minimally by controlling ATP-Mg binding.     

Dephosphorylation kinetics of pig kidney Na+,K+-ATPase

The kinetics of K+-stimulated dephosphorylation of the Na+,K+-ATPase were investigated at pH 7.4, 24 degrees C, and an ATP concentration of 1.0 mM via the stopped-flow technique using the fluorescent label RH421. Two different mixing procedures were used: (a) premixing with ATP to allow phosphorylation to go to completion, followed by mixing with KCl; and (b) simultaneous mixing with ATP and KCl. Using mixing procedure (a), the dephosphorylation rate constant of enzyme complexed with K+ ions could be determined directly to be 190 s-1).     

Phosphorylation of glycerol and dihydroxyacetone in Acetobacter xylinum and its possible regulatory role

Extracts of Acetobacter xylinum catalyze the phosphorylation of glycerol and dihydroxyacetone (DHA) by adenosine 5'-triphosphate (ATP) to form, respectively, L-alpha-glycerophosphate and DHA phosphate. The ability to promote phosphorylation of glycerol and DHA was higher in glycerol-grown cells than in glucose- or succinate-grown cells. The activity of glycerol kinase in extracts is compatible with the overall rate of glycerol oxidation in vivo. The glycerol-DHA kinase has been purified 210-fold from extracts, and its molecular weight was determined to be 50,000 by gel filtration. The glycerol kinase to DHA kinase activity ratio remained essentially constant at 1.6 at all stages of purification. The optimal pH for both reactions was 8.4 to 9.2. Reaction rates with the purified enzyme were hyperbolic functions of glycerol, DHA, and ATP. The Km for glycerol is 0.5 mM and that for DHA is 5 mM; both are independent of the ATP concentration. The Km for ATP in both kinase reactions is 0.5 mM and is independent of glycerol and DHA concentrations. Glycerol and DHA are competitive substrates with Ki values equal to their respective Km values as substrates. D-Glyceraldehyde and l-Glyceraldehyde were not phosphorylated and did not inhibit the enzyme. Among the nucleotide triphosphates tested, only ATP was active as the phosphoryl group donor. Fructose diphosphate (FDP) inhibited both kinase activities competitively with respect to ATP (Ki= 0.02 mM) and noncompetitively with respect to glycerol and DHA. Adenosine 5'-diphosphate (ADP) and adenosine 5'-monophosphate (AMP) inhibited both enzymic activities competitively with respect to ATP (Ki (ADP) = 0.4 mM; Ki (AMP) =0.25 mM). A. xylinum cells with a high FDP content did not grow on glycerol. Depletion of cellular FDP by starvation enabled rapid growth on glycerol. It is concluded that a single enzyme from A. xylinum is responsible for the phosphorylation of both glycerol and DHA. This as well as the sensitivity of the enzyme to inhibition by FDP and AMP suggest that it has a regulatory role in glycerol metabolism.     

ATP synthase. Conditions under which all catalytic sites of the F1 moiety are kinetically equivalent in hydrolyzing ATP

Conditions have been reported under which the F1 moiety of bovine heart ATP synthase catalyzes the hydrolysis of ATP by an apparently cooperative mechanism in which the slow rate of hydrolysis at a single catalytic site (unisite catalysis) is enhanced more than 10(6)-fold when ATP is added in excess to occupy one or both of the other two catalytic sites (multisite catalysis) (Cross, R. L., Grubmeyer, C., and Penefsky, H. S. (1982) J. Biol. Chem. 257, 12101-12105). In the novel studies reported here, and in contrast to the earlier report, we have (a) monitored the kinetics of ATP hydrolysis of F1 by using nucleotide-depleted preparations and a highly sensitive chemiluminescent assay; (b) followed the reaction immediately upon addition of F1 to ATP, rather than after prior incubation with ATP; and (c) used a reaction medium with Pi as the only buffer. The following observations were noted. First, regardless of the source of enzyme, bovine or rat, and catalytic conditions (unisite or multisite), the rates of hydrolysis depend on ATP concentration to the first power. Second, the first order rate constant for ATP hydrolysis remains relatively constant under both unisite and multisite conditions declining only slightly at high ATP concentration. Third, the initial rates of ATP hydrolysis exhibit Michaelis-Menten kinetic behavior with a single Vmax exceeding 100 micromol of ATP hydrolyzed per min/mg of F1 (turnover number = 635 s-1) and a single Km for ATP of about 57 microM. Finally, the reaction is inhibited markedly by low concentrations of ADP. It is concluded that, under the conditions described here, all catalytic sites that participate in the hydrolysis of ATP within the F1 moiety of mitochondrial ATP synthase function in a kinetically equivalent manner.     

Sturgeon proopiomelanocortin has a remnant of gamma-melanotropin

A thioesterase I gene was recloned and sequenced from Escherichia coli strain JM109. The overexpressed, matured enzyme from JM109 was purified to homogeneity. The enzyme showed broad hydrolytic activity toward three kinds of substrates including acyl-CoAs, esters, and amino acid derivatives. The enzyme had a kcat/Km value of 0.363 s-1 microM-1, for a typical thioesterase I substrate, palmitoyl-CoA. The arylesterase activity of the enzyme was observed by its ability to hydrolyze several aromatic esters including alpha-naphthyl acetate, alpha-naphthyl butyrate, phenyl acetate, benzyl acetate, and eight p-nitrophenyl esters. In kinetic studies a chymotrypsin-like substrate (an amino acid derivative), N-carbobenzoxy-L-phenylalanine p-nitrophenyl ester (L-NBPNPE), was the best substrate for the enzyme with a catalytic efficiency (kcat/Km) of 4.00 s-1 microM-1, which was 23 times higher than that of the enantiomer D-NBPNPE (0.171 s-1 microM-1). It was concluded that the thioesterase I of E. coli had arylesterase activity and it possessed stereospecificity for protease substrates.     

Pre-steady-state kinetic analysis of the trichodiene synthase reaction pathway

The pre-steady-state kinetics of the trichodiene synthase reaction were investigated by rapid chemical quench methods. The single-turnover rate was found to be 3.5-3.8 s-1, a rate 40 times faster than the steady-state catalytic rate (kcat = 0.09 s-1) for trichodiene synthase-catalyzed conversion of farnesyl diphosphate (FPP) to trichodiene at 15 degrees C. In a multiturnover experiment, a burst phase (kb = 4.2 s-1) corresponding to the accumulation of trichodiene on the surface of the enzyme was followed by a slower, steady-state release of products (klin = 0.086 s-1) which corresponds to kcat. These results strongly suggest that the release of trichodiene from the enzyme active site is the rate-limiting step in the overall reaction, while the consumption of FPP is the step which limits chemical catalysis at the active site. Single-turnover experiments with trichodiene synthase mutant D101E, for which the steady-state rate constant kcat is 1/3 of that of wild type, revealed that the mutation actually depresses the rate of FPP consumption by a factor of 100. The deuterium isotope effect on the consumption of [1-2H,1,2-14C]FPP was found to be 1.11 +/- 0.06. Single turnover reactions of [1,2-14C]FPP catalyzed by trichodiene synthase were carried out at 4, 15, or 30 degrees C in an effort to provide direct observation of the proposed intermediate nerolidyl diphosphate (NPP). However, no NPP was detected, indicating that the conversion of NPP must be too fast to be observed within the detection limits of the assay. Taken together, these observations suggest that the isomerization of FPP to NPP is the step which limits the rate of chemical catalysis in the trichodiene synthase reaction pathway.     

Product release is the major contributor to kcat for the hepatitis C virus helicase-catalyzed strand separation of short duplex DNA

Hepatitis C virus (HCV) helicase catalyzes the ATP-dependent strand separation of duplex RNA and DNA containing a 3' single-stranded tail. Equilibrium and velocity sedimentation centrifugation experiments demonstrated that the enzyme was monomeric in the presence of DNA and ATP analogues. Steady-state and pre-steady-state kinetics for helicase activity were monitored by the fluorescence changes associated with strand separation of F21:HF31 that was formed from a 5'-hexachlorofluorescein-tagged 31-mer (HF31) and a complementary 3'-fluorescein-tagged 21-mer (F21). kcat for this reaction was 0.12 s-1. The fluorescence change associated with strand separation of F21:HF31 by excess enzyme and ATP was a biphasic process. The time course of the early phase (duplex unwinding) suggested only a few base pairs ( approximately 2) were disrupted concertedly. The maximal value of the rate constant (keff) describing the late phase of the reaction (strand separation) was 0. 5 s-1, which was 4-fold greater than kcat. Release of HF31 from E. HF31 in the presence of ATP (0.21 s-1) was the major contributor to kcat. At saturating ATP and competitor DNA concentrations, the enzyme unwound 44% of F21:HF31 that was initially bound to the enzyme (low processivity). These results are consistent with a passive mechanism for strand separation of F21:HF31 by HCV helicase.     

Proposed steady-state kinetic mechanism for Corynebacterium ammoniagenes FAD synthetase produced by Escherichia coli

The bifunctional enzyme, FAD synthetase (FS), from Corynebacterium ammoniagenes was overproduced in Escherichia coli and purified, and its steady-state kinetic properties were investigated. Although FMN is an intermediate product in the conversion of riboflavin to FAD, FMN must be released after formation, and then rebind for adenylylation. It was shown that adenylylation of FMN is reversible; FAD and pyrophosphate can be converted to FMN and ATP by the enzyme. In contrast, under the conditions studied, phosphorylation of riboflavin is irreversible. A method is described for analysis of two catalytic cycles, occurring on one enzyme, which have a substrate and/or product in common. The binding order for the phosphorylation cycle of FS was established as riboflavin(in), ATP(in), ADP(out), and FMN(out). The order for the adenylylation cycle was ATP(in), FMN(in), pyrophosphate(out), and FAD(out). A set of steady-state constants was determined, and without additional optimization, these constants were sufficient to describe experimental progress curves for conversion of riboflavin to FAD. In independent studies, it was demonstrated that FMN binds to apo-FS with a dissociation constant of 6-7 microM, which is 2 orders of magnitude higher than the KD value for riboflavin. For the steady-state kinetic analysis, this represents reversible binding of FMN(out) in the phosphorylation cycle (cycle I), which effectively inhibits catalysis in the adenylylation cycle (cycle II).     

Enzymes harboring unnatural amino acids: mechanistic and structural analysis of the enhanced catalytic activity of a glutathione transferase containing 5-fluorotryptophan

The catalytic characteristics and structure of the M1-1 isoenzyme of rat glutathione (GSH) transferase in which all four tryptophan residues in each monomer are replaced with 5-fluorotryptophan are described. The fluorine-for-hydrogen substitution does not change the interaction of the enzyme with GSH even though two tryptophan residues (Trp7 and Trp45) are involved in direct hydrogen-bonding interactions with the substrate. The rate constants for association and dissociation of the peptide, measured by stopped-flow spectrometry, remain unchanged by the unnatural amino acid. The 5-FTrp-substituted enzyme exhibits a kcat of 73 s-1 as compared to 18 s-1 for the native enzyme toward 1-chloro-2,4-dinitrobenzene. That the increase in the turnover number is due to an enhanced rate of product release in the mutant is confirmed by the kinetics of the approach to equilibrium for binding of the product. The crystal structure of the 5-FTrp-containing enzyme was solved at a resolution of 2.0 A by difference Fourier techniques. The structure reveals local conformational changes in the structural elements that define the approach to the active site which are attributed to steric interactions of the fluorine atoms associated with 5-FTrp146 and 5-FTrp214 in domain II. These changes appear to result in the enhanced rate of product release. This structure represents the first of a protein substituted with 5-fluorotryptophan.     

The bifunctional enzyme leukotriene-A4 hydrolase is an arginine aminopeptidase of high efficiency and specificity

Leukotriene-A4 hydrolase (EC 3.3.2.6) cleaved the NH2-terminal amino acid from several tripeptides, typified by arginyl-glycyl-aspartic acid, arginyl-glycyl-glycine, and arginyl-histidyl-phenylalanine, with catalytic efficiencies (kcat/Km) > or = 1 x 10(6) M-1 s-1. This exceeds by 10-fold the kcat/Km for its lipid substrate leukotriene A4. Catalytic efficiency declined for dipeptides which had kcat/Km ratios 10-100-fold lower than tripeptides. Tetrapeptides and pentapeptides were even poorer substrates with catalytic efficiencies below 10(3) M-1 s-1. The enzyme preferentially hydrolyzed tripeptide substrates and single amino acid p-nitroanilides with L-arginine at the NH2 terminus. Peptides with proline at the second position were not hydrolyzed, suggesting a requirement for an N-hydrogen at the peptide bond cleaved. Peptides with a blocked NH2 terminus were not hydrolyzed. The specificity constant (kcat/Km) was optimal at pH 7.2 with pK values at 6.8 and 7.9; binding was maximal at pH 8.0. Serum albumins activated the peptidase, increasing tripeptide affinities (Km) by 3-10-fold and specificities (kcat/Km) by 4-13-fold. Two known inhibitors of arginine peptidases, arphamenine A and B, inhibited hydrolysis of L-arginine p-nitroanilide with dissociation constants = 2.0 and 2.5 microM, respectively. Although the primary role of LTA4 hydrolase is widely regarded as the conversion of the lipid substrate leukotriene A4 into the inflammatory lipid mediator leukotriene B4, our data are the first showing that tripeptides are "better" substrates. This is compatible with a biological role for the peptidase activity of the enzyme and may be relevant to the distribution of the enzyme in organs like the ileum, liver, lung, and brain. We present a model which accommodates the available data on the interaction of substrates and inhibitors with the enzyme. This model can account for overlap in the active site for hydrolysis of leukotriene A4 and peptide or p-nitroanilide substrates.     

Substrate specificities of pepstatin-insensitive carboxyl proteinases from gram-negative bacteria

Pseudomonas carboxyl proteinase (PCP), isolated from Pseudomonas sp. 101, and Xanthomonas carboxyl proteinase (XCP), isolated from Xanthomonas sp. T-22, are the first and second examples of unique carboxyl proteinases [EC 3.4.23.33] which are insensitive to aspartic proteinase inhibitors, such as pepstatin, diazoacetyl-DL-norleucine methylester, and 1,2-epoxy-3(p-nitrophenoxy)propane. The substrate specificities of PCP and XCP were studied using a series of synthetic chromogenic peptide substrates with the general structure, P5-P4-P3-P2-Phe-Nph-P2'-P3' (P5, P4, P3, P2, P2', P3': a variety of amino acids, Nph is p-nitro-L-phenylalanine, and the Phe-Nph bond is cleaved). PCP and XCP were shown to hydrolyze a synthetic substrate, Lys-Pro-Ala-Leu-Phe-Nph-Arg-Leu, most effectively among 28 substrates. The kinetic parameters of this peptide for PCP were Km = 6.3 microM, Kcat = 51.4 s-1, and kcat/Km = 8.16 microM-1.s-1. The kinetic parameters for XCP were Km = 3.6 microM, kcat = 52.2 s-1, and kcat/Km = 14.5 microM-1.s-1. PCP showed a stricter substrate specificity than XCP. That is, the specificity constant (kcat/Km) of each substrate for PCP was in general < 0.5 microM-1.s-1, but was drastically improved by the replacement of Lys by Leu at the P2 position. On the other hand, XCP showed a less stringent substrate specificity, with most of the peptides exhibiting reasonable kcat/Km values (> 1.0 microM-1.s-1). Thus it was found that the substrate specificities of PCP and XCP differ considerably, in spite of the high similarity in their primary structures. In addition, tyrostatin was found to be a competitive inhibitor for XCP, with a Ki value of 2.1 nM, as well as for PCP (Ki = 2.6 nM).