Initial Velocity Experiments Research Articles

Spinach nitrate reductase. Kinetic studies of NADH-diaphorase

NADH-cytochrome c reductase (diaphorase), a functional moiety of the spinach NADH-nitrate reductase complex, uses one molecule of NADH to reduce two of ferricytochrome c. Initial velocity experiments in the absence of products produce patterns of parallel lines when plotting 1/ v vs. 1/(substrate) at different fixed concentrations of the other substrate. The results agree with a Ping Pong mechanism in which the first product (NAD +) is released, with formation of a reduced free form of the enzyme, prior to binding of the first molecule of the second substrate (ferricytochrome c). Product inhibition experiments indicate the production of an abortive complex between NAD + and the oxidized enzyme form that binds NADH. The data are consistent with a Hexa Uni Ping Pong mechanism in which an abortive complex is formed between NAD + and the enzyme form binding NADH.

Studies on the mechanism of adenosine 5'-monophosphate inhibition of bovine liver fructose 1,6-bisphosphatase.

Inhibition by adenosine 5'-monophosphate (AMP) of the forward reaction of fructose 1,6-bisphosphatase (FBPase) has been studied by means of progress curve analysis. The full time course of the FBPase reaction was followed by coupling the reaction to the enzymes phosphoglucoisomerase and glucose-6-phosphate dehydrogenase. The data were analyzed by using three different methods of regression and these methods are compared. During these progress curve experiments, it was noticed that fructose 1,6-bisphosphate (Fru-P2) was not fully utilized in the presence of AMP. This anomaly could be best explained by proposing the formation of an AMP.Fur-P2 complex that was inactive with FBPase. Besides reducing the level of substrate available to FBPase, AMP caused slope-parabolic, intercept-parabolic noncompetitive inhibition with respect to Fru-P2. A kinetic model for AMP inhibition of the forward reaction of FBPase is presented. Initial rate kinetics were used to study the reverse reaction of FBPase; AMP was a slope-parabolic, intercept-parabolic noncompetitive inhibitor with respect to both fructose 6-phosphate and inorganic phosphate. Initial velocity experiments in which both fructose 6-phosphate and inorganic phosphate were varied were carried out in the absence and presence of AMP. The results of these experiments indicated to which reverse-reaction enzyme forms AMP was binding. The possible physiological significance of the AMP inhibition of FBPase and of the proposed AMP.Fru-P2 complex is discussed.

Arginyl-tRNA synthetase from Escherichia coli K12. Purification, properties, and sequence of substrate addition.

Arginyl-tRNA synthetase from Escherichia coli K12 has been purified more than 1000-fold with a recovery of 17%. The enzyme consists of a single polypeptide chain of about 60 000 molecular weight and has only one cysteine residue which is essential for enzymatic activity. Transfer ribonucleic acid completely protects the enzyme against inactivation by p-hydroxymercuriben zoate. The enzyme catalyzes the esterification of 5000 nmol of arginine to transfer ribonucleic acid in 1 min/mg of protein at 37 degrees C and pH 7.4. One mole of ATP is consumed for each mole of arginyl-tRNA formed. The sequence of substrate binding has been investigated by using initial velocity experiments and dead-end and product inhibition studies. The kinetic patterns are consistent with a random addition of substrates with all steps in rapid equilibrium except for the interconversion of the cental quaternary complexes. The dissociation constants of the different enzyme-substrate complexes and of the complexes with the dead-end inhibitors homoarginine and 8-azido-ATP have been calculated on this basis. Binding of ATP to the enzyme is influenced by tRNA and vice versa.

Biosynthesis of Glycogen in Neurospora crassa Kinetic

The kinetic mechanism of glycogen synthase [UDP-glucose: glycogen 4-alpha-glucosyltransferase, EC 2.4.1.11], glucose-6-P-dependent form, from Neurospora crassa has been investigated by initial velocity experiments and studies with inhibitors in the presence of sufficient levels of glucose-6-P. The rate equation was different from those of common two-substrate systems because one of the substrates, glycogen, is also a product. The reaction rates were determined by varying the concentration of one of the substrates while keeping that of the other constant. Double-reciprocal plots of initial velocity measurements were linear and showed converging line patterns. UDP was found to act competitively when the substrate UDP-glucose was varied, but noncompetitively when glycogen was varied. On the basis of these results, it is concluded that glycogen synthase, glucose-6-P-dependent form, from N. crassa has a rapid equilibrium random Bi-Bi mechanism. Rate constant and dissociation constants for each step of this mechanism were estimated.

Purification and properties of α-ketoadipate reductase, a newly discovered enzyme from human placenta

A newly discovered enzyme, α-ketoadipate reductase, has been purified 1000-fold from human placenta. This enzyme catalyzes the following reaction: α-ketoadipate + NADH + H + → α-hydroxyadipate + NAD. The enzyme has an estimated molecular weight of 95,000 on gel filtration and an isoelectric point at pH 7.0 on electrofocusing. Several forms of the enzyme were isolated during purification. The pH optimum for the major form was 6.3. The reaction product of α-ketoadipate reductase was identified as α-hydroxyadipate by comparison of the enzyme product with chemically prepared α-hydroxyadipate. Studies of the reaction stoichiometry indicated that equimolar quantities of NADH and α-ketoadipate were used in the synthesis of an equivalent quantity of α-hydroxyadipate. Under conditions where the remaining lactate dehydrogenase and malate dehydrogenase were completely inhibited without affecting the α-ketoadipate reductase activity, it was found that α-ketoadipate reductase was highly specific for α-ketoadipate as substrate. NADPH could not substitute for NADH. Initial velocity experiments showed that NADH was an uncompetitive substrate inhibitor.

Comparison of initial velocity and binding data for allosteric adenosine monophosphate nucleosidase.

Adensine monophosphate nucleosidase (AMP nucleosidase) from Azotobacter vinelandii is composed of six subunits with similar or identical charge and size and has a molecular weight of approximately 320,000. Binding studies with tritiated tubercidin 5' -PO4 (4-amino-7-(beta-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine-5' -monophosphate), a competitive inhibitor with respect to the substrate, AMP, indicate the presence of three independent, identical binding sites for the substrate analog. The binding of tubercidin 5' -PO4 is not affected by either Mg2+ or MgATP2-; however, in initial velocity experiments MgATP2- caused from greater than100- to 4,000-fold activation of substrate hydrolysis depending on the concentration of AMP. Binding studies with [14C]ATP are consistent with six interdependent binding sites for MgAT2-. Initial velocity and binding curves for MgATP2- are similar in shape, but reveal a disproportionate increase in initial velocity at low saturation levels of MgAT2-. Binding of MgAT2- is inhibited by increasing concentrations of P1 which acts as a competitive inhibitor of MgATP2- activation in both initial velocity and binding experiments. In the absence of MgATP2-, 32Pi binds at six or more interdependent modifier sites. The simulataneous binding of Mg[14C]ATP2- and 32Pi was studied in experiments where MgATP2- and Pi were held in constant ratio. Extrapolation to infinite concentrations of both MgATP2- and Pi indicated that 3 molecules of each were bound to the enzyme. Thus the binding of the allosteric activator and inhibitor are mutually exclusive. These results are consistent with a single modifier site per subunit at which either MgATP2- or Pi may combine, or with separate activator and inhibitor sites which cannot be filled simultaneously. Comparative initial velocity and binding studies with Pi indicate that the initial rate of AMP hydrolysis depends primarily on the extent of modifier site saturation with MgATP2-. Thus when two sites are filled with MgATP2-, the initial rate is approximately the same as when two additional modifier sites are filled with Pi. Binding of Pi, therefore, does not appear to affect the catalytic effectiveness of the active site when MgATP2- is also present, except by the displacement of MgATP2-.

Molecular and catalytic properties of ribulose 1,5-bisphosphate carboxylase from the photosynthetic extreme halophile Ectothiorhodospira halophila

D-Ribulose 1,5-bisphosphate (RuBP) carboxylase has been purified from the photosynthetic extreme halophile Ectothiorhodospira halophila. Despite a growth requirement for almost saturating sodium chloride in the medium, both crude and homogeneous preparations of RuBP carboxylase obtained from this organism were inhibited by salts. Sedimentation equilibrium analyses showed the enzyme to be large (molecular weight: 601,000). The protein was composed of two types of polypeptide chains of 56,000 and of 18,000 daltons. The small subunit appeared to be considerably larger than the small subunit obtained from the RuBP carboxylase isolated from Chromatium, an organism related to E. halophila. Amino acid analyses of hydrolysates of both E. halophilia and Chromatium RuBP carboxylases were very similar. Initial velocity experiments showed that the E. halophila RuBP carboxylase had a Km for ribulose diphosphate of 0.07 mM and a Km for HCO3- of 10 mM. Moreover, 6-phospho-D-gluconate was found to markedly inhibit the E. halophila carboxylase; a Ki for phosphogluconate of 0.14 mM was determined.

Kinetic Characteristics of the Pyridine Nucleotide Transhydrogenase from Pseudomonas aeruginosa

Abstract Kinetic studies on Pseudomonas aeruginosa pyridine nucleotide transhydrogenase yield the following information. In the presence of 5 x 10-4 m 2'-AMP the initial velocity experiments for both the TPNH-(TN)DPN+ (oxidized thionicotinamide DPN) and DPNH-(TN)DPN+ reactions are consistent with a bi bi catalytic reaction mechanism. DPN+ inhibition of the DPNH-(TN)DPN+ reaction is the type expected for this mechanism. When 2'-AMP is not included in the TPNH-(TN)DPN+ system the reaction velocity shows a second order dependence on TPNH, but a first order dependence on (TN)DPN+. The maximum velocity for the TPNH-(TN)DPN+ system is apparently the same in the presence as in the absence of 5 x 10-4 m 2'-AMP. However, in the presence of 5 x 10-4 m 2'-AMP the reaction velocity shows a first order dependence on both substrates. These experiments show that both TPNH and 2'-AMP are activators of the enzyme. In the DPNH-(TN)DPN+ reaction the velocity shows a first order dependence on both substrates and a second order dependence on 2'-AMP. 2'-AMP causes a decrease in the apparent Michaelis constant for DPNH and an increase in the apparent maximum velocity. If the mechanism is assumed to be ping-pong bi bi then the experiments indicate that 2'-AMP increases the rates of steps in the portion of the reaction sequence involving reduction of the enzyme by DPNH. Experiments indicate that TPN+ does not have the same capacity for activation as do TPNH and 2'-AMP. In contrast to DPN+, TPN+ at low concentrations is competitive with DPNH in the DPNH-(TN)DPN+ reaction. Thus failure of the transhydrogenase to catalyze the reaction between DPNH and TPN+ can be explained by the failure of TPN+ to cause full activation, making DPNH a less effective substrate, and also by formation by TPN+ of a dead end complex with enzyme.

The Anthranilate Synthetase-Anthranilate 5-Phosphoribosylpyrophosphate Phosphoribosyltransferase Aggregate: PURIFICATION OF THE AGGREGATE AND REGULATORY PROPERTIES OF ANTHRANILATE SYNTHETASE

Abstract Relatively stable, highly purified preparations of the anthranilate synthetase-anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase aggregate have been obtained from Salmonella tryphimurium. A molecular weight of approximately 285,000 was estimated for the enzyme aggregate by sucrose gradient centrifugation. Anthranilate synthetase activity was completely inhibited by 10 µm tryptophan under appropriate conditions. Inhibition by tryptophan was competitive with chorismate and noncompetitive with glutamine and showed positive cooperativity. Anthranilate synthetase activity was almost completely insensitive to inhibition by tryptophan in the presence of 5.0 mm Mg2+, suggesting distinct substrate and inhibitor sites. Mg2+ was required for enzymatic activity. Inhibition of enzyme activity was obtained by low concentrations of anthranilate, indicating possible regulatory significance. This inhibition was partially overcome by excess Mg2+, suggesting that inhibition was due, at least in part, to interaction of anthranilate with the tryptophan regulatory site. Positive cooperativity obtained for anthranilate was similar to that obtained for tryptophan. In the presence of anthranilate or tryptophan positive cooperativity for chorismate was observed. For the NH3-dependent anthranilate synthetase activity of the anthranilate synthetase-anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase aggregate, negative cooperativity for NH3 was indicated by the shapes of plots of initial velocity against (NH4)2SO4 concentration and Lineweaver-Burk plots and the values for related kinetic constants. Initial velocity experiments were consistent with a sequential mechanism.

Kinetic Mechanism of Phosphorylase b: RATES OF INITIAL VELOCITIES AND OF ISOTOPE EXCHANGE AT EQUILIBRIUM

In order to assign a kinetic mechanism to phosphorylase b, initial rate studies were conducted in conjunction with isotope exchange studies at equilibrium. Initial velocity rates were measured with varied concentrations of both substrates in each direction, in the presence of saturating levels of AMP. Data were analyzed with double reciprocal plots and secondary replots of intercepts and slopes. The resulting kinetically derived dissociation constants agreed reasonably well with those determined by independent means. These results indicate that the kinetic mechanism of phosphorylase b is rapid equilibrium random bi bi in nature. The rate equation for this mechanism has been modified to take into account the fact that one of the substrates, glycogen, gives rise to a product which is chemically and kinetically indistinguishable under the conditions used to determine initial rates. This phenomenon, equivalent to having one of the two products present at all times, results in the number two being introduced into the rate equation, so the kinetically derived dissociation constant for glycogen is half the true value while the observed Km values for phosphate and glucose 1-phosphate are twice the theoretical values. This rate equation should apply wherever an enzyme synthesizes or degrades a homopolymer by 1 unit at a time. To confirm the mechanism suggested by initial velocity experiments, isotope exchange studies at equilibrium were performed for the phosphorylase b system, again in the presence of saturating levels of AMP. The 14C-glucose-1-P ⇌ glycogen equilibrium reaction rate increased as the concentrations of either glucose-1-P and orthophosphate or glycogen were increased, and reached a plateau as the concentration of varied substrates became saturating. The same results were obtained for 32Pi ⇌ glucose-1-P exchange. Thus, there was no evidence of an inhibition of the exchange of one pair of substrates when the concentration of the other substrate pair was raised. Similar exchange rates were observed in either direction, indicating that rapid equilibrium conditions apply. A reasonable agreement existed between the maximal velocities calculated from the initial rate data and those determined from the isotope exchange rates, assuming a rapid equilibrium random bi bi mechanism. Both the initial velocity and the equilibrium isotope exchange studies support a reaction mechanism in which substrates bind in a noncompulsory order to the enzyme and in which the interconversions of ternary complexes are the ratelimiting steps.

Comments on the kinetics and mechanism of yeast hexokinase action. Is the binding sequence of substrates to the enzyme ordered or random?

The mechanism of action of yeast hexokinase was reinvestigated in light of a recent report in which it was suggested that substrates add to the enzyme in an ordered manner with the binding of glucose required to precede the binding of ATP. Initial velocity experiments were undertaken with AMP which is a competitive inhibitor for ATP, and with the product inhibitor glucose‐6‐phosphate. The results of these studies serve to exclude the ordered mechanism for yeast hexokinase from consideration. A discussion is presented in an attempt to show that yeast hexokinase may react in a random fashion with its substrates before forming a ternary complex of enzyme‐ATP‐glucose.

Coenzyme A-linked Aldehyde Dehydrogenase from Escherichia coli: I. PARTIAL Purification, Properties, and Kinetic Studies of the Enzyme

Abstract Coenzyme A-linked aldehyde dehydrogenase from Escherichia coli strain B was purified 170-fold over cell-free extracts, and certain of its properties were investigated. The enzyme is essentially inactive in the absence of sulfhydryl compounds such as dithiothreitol and β-mercaptoethanol. Addition of a thiol reagent after incubation of the enzyme with substrates results in very slow reactivation. Incubation of aldehyde dehydrogenase with DPN plus mercaptan before addition of other substrates is required in order to obtain normal initial velocities. The kinetics of the system was investigated from both sides of the reaction. Initial velocity experiments suggest the mechanism is ping-pong. These studies, although still preliminary, imply that acetyl-CoA adds to the dehydrogenase before addition of DPNH. With regard to the other side of the reaction, it is thought that DPN and acetaldehyde interact with the enzyme prior to the addition of CoA. The last step in the reaction sequence may be a transacetylation between the enzyme and CoA to form acetyl-CoA. Additional properties of the enzyme, such as optimum pH, substrate specificity, and reaction stoichiometry, were also studied.