Identification of the catalytic mechanism and estimation of kinetic parameters for fumarase

The enzyme fumarase catalyzes the reversible hydration of fumarate to malate. The reaction catalyzed by fumarase is critical for cellular energetics as a part of the tricarboxylic acid cycle, which produces reducing equivalents to drive oxidative ATP synthesis. A catalytic mechanism for the fumarase reaction that can account for the kinetic behavior of the enzyme observed in both isotope exchange studies and initial velocity studies has not yet been identified. In the present study, we develop an 11-state kinetic model of the enzyme based on the current consensus on its catalytic mechanism and design a series of experiments to estimate the model parameters and identify the major flux routes through the mechanism. The 11-state mechanism accounts for competitive binding of inhibitors and activation by different anions, including phosphate and fumarate. The model is identified from experimental time courses of the hydration of fumarate to malate obtained over a wide range of buffer and substrate concentrations. Further, the 11-state model is found to effectively reduce to a five-state model by lumping certain successive steps together to yield a mathematically less complex representation that is able to match the data. Analysis suggests the primary reaction route of the catalytic mechanism, with fumarate binding to the free unprotonated enzyme and a proton addition prior to malate release in the fumarate hydration reaction. In the reverse direction (malate dehydration), malate binds the protonated form of the enzyme, and a proton is generated before fumarate is released from the active site.     

Kinetics of carbonic anhydrase catalysis in solvents of increased viscosity: a partially diffusion-controlled reaction

Steady-state kinetic studies of the bovine carbonic anhydrase B-catalyzed hydration of CO2, dehydration of HCO3-, and hydrolysis of p-nitrophenylacetate were made in glycerol/water solvents of increased viscosity in order that the effect of diffusion-control on the substrate association reactions could be determined. The minimum association rate constants (kmin = V/(Km[E0])) were obtained at low substrate concentrations. The esterase activity did not depend upon the solvent viscosity. However, both the CO2 hydration and HCO3- dehydration reactions depended upon the solvent viscosity consistent with partial diffusion control. Thus both chemical activation and diffusion control processes contribute to the observed kmin. In low-viscosity aqueous solutions both hydration and dehydration are largely controlled by chemical activation. However, at higher viscosities, equal to that found in the interior of the erythrocyte, both reactions are largely diffusion controlled. This result can be interpreted to mean that carbonic anhydrase is a highly evolved enzyme that has approached its maximum efficiency. The extent of diffusion control observed rules out H2CO3 as a significant reactant with the enzyme. Several models that yield minimum steric requirements for access of substrate to the active site are examined. Minimum steric constraints are less for the smaller CO2. The slower esterase reaction is not influenced by diffusion.     

Energetic limits of phosphotransfer in the catalytic subunit of cAMP-dependent protein kinase as measured by viscosity experiments.

Viscosogenic agents were used to test the diffusion limits of the reaction catalyzed by the catalytic subunit of the cAMP-dependent protein kinase. The effects of glycerol and sucrose on the maximum rate (kcat) and the apparent second-order rate constants (kcat/Kpeptide) for the phosphorylation of four peptidic substrates were measured at their pH optima. The agents were found to have moderate to no effect on kcat/Kpeptide for good and poor substrates, respectively. Conversely, kcat was highly sensitive to solvent viscosity for three of the four peptides at high concentrations of ATP. Taken together, these data indicate that enzymatic phosphorylation by the catalytic subunit proceeds with rapid or near rapid equilibrium binding of substrates and that all steps following the central substrate complex (i.e., chemical and conformational events) are fast relative to the rate-determining dissociation of product, ADP, when ATP levels are high. Under saturating concentrations of peptide I, LRRASLG, an unproductive form of the enzyme is populated. The observed phosphorylation rate from this complex is involved in rate limitation owing to a slow step separating unproductive and productive enzyme forms. The data are used to establish a kinetic mechanism for the catalytic subunit that predicts initial reaction velocities under varying concentrations of ATP and substrate.     

Chicken fumarase. II. Kinetic studies.

The catalysis of the hydration of fumarate and deshydration of L - malate by chicken fumarase was measured spectrophotometrically over a range of substrate concentrations from 4 times 10(-3) M to 8 times 10(-5) M for fumarate and from 8 times 10(-2) M to 10(-3) M for L - malate. For the forward and reverse reactions, linear Lineweaver and Burk plots were obtained. The Michaelis constants and the maximum initial velocities for both substrates were determined and the Haldane relation was found to be obeyed. The effect of pH on activity was investigated over a pH range from 5.5 to 9.0 and the data indicate the presence, in the active site, of two ionizable groups, one in the acidic form and one in the basic form. The values of the ionization constants, determined for the enzyme - substrate complexes, agree closely with the ones obtained for the porcine enzyme. The mode of action of twenty-four structural analogs on the initial velocity of the dehydration of L-malate, by chicken fumarase was examined. From these studies, two regions positively charged appear necessary for the effective binding of the carboxylates of the substrates and competitive inhibitors to the active center. Moreover, the data suggest the presence of an additional group, in the catalytic site of chicken fumarase, that stabilizes the carbon-carbon double bond common to fumarate and its structural analogs. Finally, from the comparison of the kinetic properties of the chicken and pig fumarases, it may be concluded that the catalytic mechanism of the homologous enzymes are very similar, if not identical.     

Mechanism of activation of the NAD-malic enzyme from Ascaris suum by fumarate.

The mechanism of activation of the NAD-malic enzyme from Ascaris suum by fumarate has been probed using initial velocity studies, deuterium isotope effects, and isotope partitioning of the E:Mg:malate complex. Fumarate exerts its activating effect by decreasing the off-rate for malate from the E:Mg:malate and E:NAD:Mg:malate complexes. Fumarate is a positive heterotropic effector of the NAD-malic enzyme at low concentrations (K act approximately 0.05 mM) and an inhibitor competitive against malate (Ki approximately 25 mM). The activation by fumarate results in a decrease in the Ki malate and an increase in V/K malate of about 2-fold, while the maximum velocity remains constant. Isotope partitioning studies of E:Mg:[14C]malate indicate that the presence of fumarate results in a decrease in the malate off-rate constant by about 2.2-fold. The deuterium isotope effects on V and V/K malate are both 1.6 +/- 0.1 in the absence of fumarate, while in the presence of 0.5 mM fumarate DV is 1.6 +/- 0.1 and D(V/K malate) is 1.1 +/- 0.1. These data are also consistent with a decrease in the off-rate for malate from E:NAD:Mg:malate, resulting in an increase in the forward commitment factor for malate and manifested as a lower value for D(V/K malate). There is a discrimination between active and activator sites for the binding of dicarboxylic acids, with the activator site preferring the extended configuration of 4-carbon dicarboxylic acids, while the active site prefers a configuration in which the 4-carboxyl is twisted out of the C1-C3 plane. The physiologic importance and regulatory properties of fumarate in the parasite are also discussed.     

Ascaris suum NAD-malic enzyme is activated by L-malate and fumarate binding to separate allosteric sites

The kinetic mechanism of activation of the mitochondrial NAD-malic enzyme from the parasitic roundworm Ascaris suum has been studied using a steady-state kinetic approach. The following conclusions are suggested. First, malate and fumarate increase the activity of the enzyme in both reaction directions as a result of binding to separate allosteric sites, i.e., sites that exist in addition to the active site. The binding of malate and fumarate is synergistic with the K(act) decreasing by >or=10-fold at saturating concentrations of the other activator. Second, the presence of the activators decreases the K(m) for pyruvate 3-4-fold, and the K(i) (Mn) >or=20-fold in the direction of reductive carboxylation; similar effects are obtained with fumarate in the direction of oxidative decarboxylation. The greatest effect of the activators is thus expressed at low reactant concentrations, i.e., physiologic concentrations of reactant, where activation of >or=15-fold is observed. A recent crystallographic structure of the human mitochondrial NAD malic enzyme [13] shows fumarate bound to an allosteric site. Site-directed mutagenesis was used to change R105, homologous to R91 in the fumarate activator site of the human enzyme, to alanine. The R105A mutant enzyme exhibits the same maximum rate and V/K(NAD) as does the wild-type enzyme, but 7-8-fold decrease in both V/K(malate) and V/K(Mg), indicating the importance of this residue in the activator site. In addition, neither fumarate nor malate activates the enzyme in either reaction direction. Finally, a change in K143 (a residue in a positive pocket adjacent to that which contains R105), to alanine results in an increase in the K(act) for malate by about an order of magnitude such that it is now of the same magnitude as the K(m) for malate. The K143A mutant enzyme also exhibits an increase in the K(act) for fumarate (in the absence of malate) from 200 microM to about 25 mM.     

Proton transfer in catalysis by fumarase.

Using 3T[14C]malate it was possible to show intermolecular T-transfer to unlabeled fumarate. The rate of dissociation of ET derived from the malate was not rapid, only about as fast as required for KMcat. Because of the slow dissociation of ET derived from T-malate, the awkward complex ET-malate is readily formed. As shown by the effect of added malate on the partition of ET, otherwise captured by fumarate, ET.malate must be functional. Its rate of dissociation to E.M determines the V/Km value of malate. Hydrogen dissociation of the complex ET.F was linearly related to the concentration and basicity of the buffer provided, varying from < 10% to > 60% of the overall rate with alkyl phosphonates. Partition of EH.F to free malate or fumarate occurs in a ratio approximately 2:1 at both low and high buffer. This agrees well with the comparison of the equilibrium exchange rates: malate with [18O]water to malate with [14C]-fumarate [Hansen, J.N., Dinovo, E.C., & Boyer, P.D. (1969) J. Biol. Chem. 244, 6270-6279]. Therefore, the abstracted hydroxyl group is fully exchanged from the enzyme when the bound hydrogen and fumarate return to malate and must be much more accessible to the medium than the abstracted proton. The fact that buffer increases the rate of proton transfer to the medium in the central complex makes it appear that a proton relay connects the active site donor with a remote site that interfaces with the ultimate proton source, water.     

Enzyme kinetics in solvents of increased viscosity. Dynamic aspects of carbonic anhydrase catalysis

The dependence of enzymatic catalysis on diffusion rates in solution was examined with regard to high specific activity carbonic anhydrase (CA II) by varying the viscosity of the reaction medium with added glycerol, sucrose, and ficoll (a copolymer of sucrose and epichlorohydrin). Responses of the Michaelis-Menten parameters associated with CO2 hydration and HCO3- dehydration were deduced and analyzed by utilizing a spectrophotometric stopped-flow technique. It was found that both kcatHCO3 (= 3.9 X 10(5) s-1 at pH 5.90) and kcatCO2 (= 1.2 X 10(5) s-1 at pH 5.90 and 8.6 X 10(5) s-1 at pH 8.80) steadily decreased with the addition of monomeric viscogen while both KmHC03- (= 20 mM at pH 5.90) and KmCO2 (= 18 mM at pH 5.90 and 13 mM at pH 8.80) remained independent of viscosity, within experimental error. These results indicate that some kind of proton-transfer-related event is primarily responsible for the observed rate decrease. The three polyhydroxy cosolutes exhibited significant differences with regard to the magnitude of the viscosity effect on the kcat of the enzyme, with glycerol affecting the largest decrease, sucrose affecting a moderate one, and ficoll having virtually no effect. The discrepancy between glycerol and sucrose could be largely reconciled by correcting for diffusion-unrelated effects as estimated from rate studies of considerably slower CA II catalyzed acetaldehyde hydration and p-nitrophenyl acetate hydrolysis. Ficoll, however, was found to be unsuitable as a viscogenic probe because it failed to appreciably decrease the mobilities of smaller ions (as deduced from electrolytic conductance measurements) despite its capacity to greatly increase the macroscopic viscosity of the medium. Our best estimates indicate that this reaction comes within ca. 30% of the diffusion limit at 0.890 cP and 25 degrees C for both CO2 hydration and HCO3- dehydration reactions. However, it is reasonable to expect this value to be considerably higher in the natural environment of the enzyme because of the relatively high viscosities attained in the interior of erythrocytes.     

A method for the isolation of Chinese hamster cell variants with an altered ability to utilise carbohydrates

Chinese hamster ovary cells (CHO-K1) are able to utilise only a few carbohydrates for growth such as glucose, mannose, fructose and galactose. They do not grow on ribose, lactose, sucrose, glycerol, lactate, pyruvate, citrate, succinate, fumarate or malate nor on glycogenic or ketogenic amino acids. After mutagenesis and selection in glucose free medium supplemented with various, individual, growth substrates, we have isolated single-cell derived clones which are now able to grow on one of the following energy source: ribose, lactose, sucrose or lactate.     

Kinetics of phloretin binding to phosphatidylcholine vesicle membranes

The submillisecond kinetics for phloretin binding to unilamellar phosphatidylcholine (PC) vesicles was investigated using the temperature-jump technique. Spectrophotometric studies of the equilibrium binding performed at 328 nm demonstrated that phloretin binds to a single set of independent, equivalent sites on the vesicle with a dissociation constant of 8.0 microM and a lipid/site ratio of 4.0. The temperature of the phloretin-vesicle solution was jumped by 4 degrees C within 4 microseconds producing a monoexponential, concentration-dependent relaxation process with time constants in the 30--200-microseconds time range. An analysis of the concentration dependence of relaxation time constants at pH 7.30 and 24 degrees C yielded a binding rate constant of 2.7 X 10(8) M-1 s-1 and an unbinding constant of 2,900 s-1; approximately 66 percent of total binding sites are exposed at the outer vesicle surface. The value of the binding rate constant and three additional observations suggest that the binding kinetics are diffusion limited. The phloretin analogue, naringenin, which has a diffusion coefficient similar to phloretin yet a dissociation constant equal to 24 microM, bound to PC vesicle with the same rate constant as phloretin did. In addition, the phloretin-PC system was studied in buffers made one to six times more viscous than water by addition of sucrose or glycerol to the differ. The equilibrium affinity for phloretin binding to PC vesicles is independent of viscosity, yet the binding rate constant decreases with the expected dependence (kappa binding alpha 1/viscosity) for diffusion-limited processes. Thus, the binding rate constant is not altered by differences in binding affinity, yet depends upon the diffusion coefficient in buffer. Finally, studies of the pH dependence of the binding rate constant showed a dependence (kappa binding alpha [1 + 10pH-pK]) consistent with the diffusion-limited binding of a weak acid.     

Adenosine deaminase: viscosity studies and the mechanism of binding of substrate and of ground- and transition-state analogue inhibitors

We have studied the effects of viscosogenic agents, sucrose and ficoll, on (1) the hydrolysis of adenosine and of 6-methoxypurine riboside catalyzed by adenosine deaminase and (2) the rates of association and dissociation of ground-state and transition-state analogue inhibitors. For adenosine, Vmax/Km is found to be inversely proportional to the relative viscosity with sucrose, an agent affecting the microscopic viscosity, while no effect is found with ficoll, an agent affecting the macroscopic viscosity. Viscosogenic agents have no effect on the kinetic constants for 6-methoxypurine riboside. Thus, the bimolecular rate constant, Vmax/Km = 11.2 +/- 0.8 microM-1 s-1, for the reaction with adenosine is found to be at the encounter-controlled limit while that for the reaction with the poor substrate 6-methoxypurine riboside, 0.040 +/- 0.004 microM-1 s-1, is limited by some other process. Viscosity-dependent processes do not make a significant (less than 10%) contribution to Vmax. The dissociation constants for inhibitors are unaffected by viscosity. The ground-state analogue inhibitor purine riboside appears to bind at a rate comparable to that of adenosine. However, the slower rates of association (0.16-2.5 microM-1 s-1) and dissociation (5 X 10(-6) to 12 s-1) of transition-state analogue inhibitors are affected by the viscosity of the medium to approximately the same extent as the encounter-controlled rates of association and dissociation of adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the purified hyaluronan synthase from Streptococcus equisimilis

Hyaluronan synthase (HAS) utilizes UDP-GlcUA and UDP-GlcNAc in the presence of Mg(2+) to form the GAG hyaluronan (HA). The purified HAS from Streptococcus equisimilis (seHAS) shows high fidelity in that it only polymerizes the native substrates, UDP-GlcNAc and UDP-GlcUA. However, other uridinyl nucleotides and UDP-sugars inhibited enzyme activity, including UDP-GalNAc, UDP-Glc, UDP-Gal, UDP-GalUA, UMP, UDP, and UTP. Purified seHAS was approximately 40% more active in 25 mM, compared to 50 mM, PO(4) in the presence of either 50 mM NaCl or KCl, and displayed a slight preference for KCl over NaCl. The pH profile was surprisingly broad, with an effective range of pH 6.5-11.5 and the optimum between pH 9 and 10. SeHAS displayed two apparent pK(a) values at pH 6.6 and 11.8. As the pH was increased from approximately 6.5, both K(m) and V(max) increased until pH approximately 10.5, above which the kinetic constants gradually declined. Nonetheless, the overall catalytic constant (120/s) was essentially unchanged from pH 6.5 to 10.5. The enzyme is temperature labile, but more stable in the presence of substrate and cardiolipin. Purified seHAS requires exogenous cardiolipin for activity and is very sensitive to the fatty acyl composition of the phospholipid. The enzyme was inactive or highly activated by synthetic cardiolipins containing, respectively, C14:0 or C18:1(Delta9) fatty acids. The apparent E(act) for HA synthesis is 40 kJ (9.5 kcal/mol) disaccharide. Increasing the viscosity by increasing concentrations of PEG, ethylene glycol, glycerol, or sucrose inhibited seHAS activity. For PEGs, the extent of inhibition was proportional to their molecular mass. PEGs with average masses of 2.7, 11.7, and 20 kg/mol caused 50% inhibition of V(max) at 21, 6.5, and 3.5 mM, respectively. The apparent K(i) values for ethylene glycol, glycerol, and sucrose were, respectively, 4.5, 3.3, and 1.2 mM.     

Linkage of EcoRI dissociation from its specific DNA recognition site to water activity, salt concentration, and pH: separating their roles in specific and non-specific binding.

We have measured the dependencies of both the dissociation rate of specifically bound EcoRI endonuclease and the ratio of non-specific and specific association constants on water activity, salt concentration, and pH in order to distinguish the contributions of these solution components to specific and non-specific binding. For proteins such as EcoRI that locate their specific recognition site efficiently by diffusing along non-specific DNA, the specific site dissociation rate can be separated into two steps: an equilibrium between non-specific and specific binding of the enzyme to DNA, and the dissociation of non-specifically bound protein. We demonstrated previously that the osmotic dependence of the dissociation rate is dominated by the equilibrium between specific and non-specific binding that is independent of the osmolyte nature. The remaining osmotic sensitivity linked to the dissociation of non-specifically bound protein depends significantly on the particular osmolyte used, indicating a change in solute-accessible surface area. In contrast, the dissociation of non-specifically bound enzyme accounts for almost all the pH and salt-dependencies. We observed virtually no pH-dependence of the equilibrium between specific and non-specific binding measured by the competition assay. The observed weak salt-sensitivity of the ratio of specific and non-specific association constants is consistent with an osmotic, rather than electrostatic, action. The seeming lack of a dependence on viscosity suggests the rate-limiting step in dissociation of non-specifically bound protein is a discrete conformational change rather than a general diffusion of the protein away from the DNA.     

Binary and ternary complexes of malate dehydrogenase with substrates and substrate analogs

Hydroxypyrenetrisulfonate binds to pig mitochondrial malate dehydrogenase (L-malate: NAD+ oxidoreductase, EC 1.1.1.37) in the presence and absence of coenzymes with a stoichiometry of one dye molecule/enzyme subunit. Binding is competitive with substrates and known substrate analogs as well as with squaric acid, a newly detected analog forming a ternary complex with enzyme/NAD+ similar to enzyme/NAD+/sulfite. Displacement of hydroxypyrenetrisulfonate by substrates and analogs was used to determine dissociation constants of binary and ternary complexes. Binary complexes form with dissociation constants of about 10 mM. They may be important for kinetic studies at high substrate concentrations where oxaloacetate inhibition and malate activation have been described.     

Electrogenic partial reactions of the gastric H,K-ATPase

The fluorescent styryl dye RH421 was used to identify and investigate electrogenic reaction steps of the H,K-ATPase pump cycle. Equilibrium titration experiments were performed with membrane vesicles isolated from hog gastric mucosa, and cytoplasmic and luminal binding of K(+) and H(+) ions was studied. It was found that the binding and release steps of both ion species in both principal conformations of the ion pump, E(1) and P-E(2), are electrogenic, whereas the conformation transitions do not contribute significantly to a charge movement within the membrane dielectric. This behavior is in agreement with the transport mechanism found for the Na,K-ATPase and the sarcoplasmic reticulum Ca-ATPase. The data were analyzed on the basis of the Post-Albers reaction cycle. For proton binding, two pK values were found in both conformations: 6.7 and 相似文献   

Protonation mechanism and location of rate-determining steps for the Ascaris suum nicotinamide adenine dinucleotide-malic enzyme reaction from isotope effects and pH studies

The pH dependence of the kinetic parameters and the primary deuterium isotope effects with nicotinamide adenine dinucleotide (NAD) and also thionicotinamide adenine dinucleotide (thio-NAD) as the nucleotide substrates were determined in order to obtain information about the chemical mechanism and location of rate-determining steps for the Ascaris suum NAD-malic enzyme reaction. The maximum velocity with thio-NAD as the nucleotide is pH-independent from pH 4.2 to 9.6, while with NAD, V decreases below a pK of 4.8. V/K for both nucleotides decreases below a pK of 5.6 and above a pK of 8.9. Both the tartronate pKi and V/Kmalate decrease below a pK of 4.8 and above a pK of 8.9. Oxalate is competitive vs. malate above pH 7 and noncompetitive below pH 7 with NAD as the nucleotide. The oxalate Kis increases from a constant value above a pK of 4.9 to another constant value above a pK of 6.7. The oxalate Kii also increases above a pK of 4.9, and this inhibition is enhanced by NADH. In the presence of thio-NAD the inhibition by oxalate is competitive vs. malate below pH 7. For thio-NAD, both DV and D(V/K) are pH-independent and equal to 1.7. With NAD as the nucleotide, DV decreases to 1.0 below a pK of 4.9, while D(V/KNAD) and D(V/Kmalate) are pH-independent. Above pH 7 the isotope effects on V and the V/K values for NAD and malate are equal to 1.45, the pH-independent value of DV above pH 7. From the above data, the following conclusions can be made concerning the mechanism for this enzyme. Substrates bind to only the correctly protonated form of the enzyme. Two enzyme groups are necessary for binding of substrates and catalysis. Both NAD and malate are released from the Michaelis complex at equal rates which are equal to the rate of NADH release from E-NADH above pH 7. Below pH 7 NADH release becomes more rate-determining as the pH decreases until at pH 4.0 it completely limits the overall rate of the reaction.     

Thermodynamics of the conversion of fumarate to L-(-)-malate

The thermodynamics of the conversion of aqueous fumarate to L-(-)-malate has been investigated using both heat conduction microcalorimetry and a gas chromatographic method for determining equilibrium constants. The reaction was carried out in aqueous Tris-HCl buffer over the pH range 6.3-8.0, the temperature range 25-47 degrees C, and at ionic strengths varying from 0.0005 to 0.62 mol kg-1. Measured enthalpies and equilibrium ratios have been adjusted to zero ionic strength and corrected for ionization effects to obtain the following standard state values for the conversion of aqueous fumarate 2- to malate 2- at 25 degrees C: K = 4.20 +/- 0.05, delta G degrees = -3557 +/- 30 J mol-1, delta H degrees = -15670 +/- 150 J mol-1, and delta C degrees p = -36 +/- J mol-1 K-1. Equations are given which allow one to calculate the combined effects of pH and temperature on equilibrium constants and enthalpies of this reaction.     

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

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

Development of defined and minimal media for the growth of Bacillus stearothermophilus.

Defined media, both solid and liquid, that support good growth of Bacillus stearothermophilus 1503 have been developed. Data are presented which indicate that manganese is required at relatively high concentrations for growth in a defined liquid medium. Phosphate concentrations higher than 5 times 10(-3) M have been shown to inhibit colony formation on solid media. Maximum viable counts of approximately 10(9) colony-forming units per ml were obtained in both the defined and minimal liquid media. Glucose, fructose, sucrose, glycerol, and starch support the growth of this obligate thermophile in the defined media, whereas citrate, alpha-ketoglutarate, succinate, fumarate, malate, acetate, and lactate do not. The described media have been utilized to isolate several amino acid-requiring mutants of B. stearothermophilus.