Rapid Equilibrium Random Bi-bi Mechanism Research Articles

Kinetics and Regulation of Mammalian NADH-Ubiquinone Oxidoreductase (Complex I)

NADH-ubiquinone oxidoreductase (Complex I, European Commission No. 1.6.5.3) is one of the respiratory complexes that generate the proton-motive force required for the synthesis of ATP in mitochondria. The catalytic mechanism of Complex I has not been well understood, due to the complicated structure of this enzyme. Here, we develop a kinetic model for Complex I that accounts for electron transfer from NADH to ubiquinone through protein-bound prosthetic groups, which is coupled to the translocation of protons across the inner mitochondrial membrane. The model is derived based on the tri-bi enzyme mechanism combined with a simple model of the conformational changes associated with proton transport. To study the catalytic mechanism, parameter values are estimated by analyzing kinetic data. The model is further validated by independent data sets from additional experiments, effectively explaining the effect of pH on enzyme activity. Results imply that matrix pH significantly affects the enzyme turnover processes. The overall kinetic analysis demonstrates a hybrid ping-pong rapid-equilibrium random bi-bi mechanism, consolidating the characteristics from previously reported kinetic mechanisms and data.

Purine nucleoside phosphorylase from bovine lens: purification and properties

Purine nucleoside phosphorylase (purine nucleoside: orthophosphate ribosyltransferase, EC 2.4.2.1.) was purified 38 750-fold to apparent electrophoretic homogeneity from bovine ocular lens. The enzyme appears to be a homotrimer with a molecular weight of 97 000, and displays non-linear kinetics with concave downward curvature in double-reciprocal plots with orthophosphate as variable substrate. The analysis of the kinetic parameters of bovine lens purine nucleoside phosphorylase, determined both for the phosphorolytic activity on nucleosides and for ribosylating activity on purine bases, indicates the occurrence of a rapid equilibrium random Bi-Bi mechanism with formation of abortive complexes. The effect of pH on the enzyme activity and on the sensitivity of the enzyme to photoinactivation, as well as the effect of thiol reagents on the enzyme activity and stability, strongly suggest the involvement of histidine and cysteine residues in the active site. From the measurements of the kinetic parameters at different temperatures, heats of formation of the enzyme-substrate complex for guanosine, guanine, orthophosphate and ribose 1-phosphate were determined. Activation energies of 15 250 and 14 650 cal/mol were obtained for phosphorolysis and synthesis of guanosine, respectively.

Purification and kinetic and structural properties of spinach leaf NADP-dependent nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase

NADP-dependent nonphosphorylating d-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.9) from spinach leaves has been purified to apparent electrophoretic homogeneity by ammonium sulfate fractionation, molecular sieving on Sephadex G-200, DEAE-cellulose, and 2′,5′-ADP-Sepharose affinity chromatography. The purified enzyme exhibited a specific activity of 15 μmol (mg protein) −1 min −1 and was characterized as a homotetramer with a native molecular weight of 195,000. Preincubation of the purified enzyme with NADP + resulted in an almost twofold increase in enzymatic activity. The rate of activation was slower than the rate of catalysis, indicating that the enzyme has hysteretic properties. This behavior results in a lag phase during activity measurement of the enzyme preincubated without NADP +. Substrate interaction and product inhibition studies suggest a rapid equilibrium random BiBi mechanism for the reaction. Thiol modifying reagents, iodoacetamide and diamide, completely inactivated the purified enzyme. Inactivation by iodoacetamide exhibited pseudo-first-order kinetics with a rate constant of 0.17 min −1. d-Glyceraldehyde 3-phosphate effectively protected the enzyme against inactivation by thiol reagents, suggesting that modification occurred at or near the substrate-binding site. Complete inactivation of the dehydrogenase was correlated with incorporation of 8 mol [1- 14C]iodoacetaniide/mol enzyme. Total protection afforded by d-glyceraldehyde 3-phosphate against enzyme inactivation by iodoacetamide was correlated with a protection of 4 mol reactive residues/mol enzyme. On the basis of these results it is suggested that one sulfhydryl group per enzyme subunit is essential for catalysis in spinach leaf nonphosphorylating glyceral-dehyde-3-phosphate dehydrogenase. A kinetic and molecular mechanism for the reaction is proposed.

Kinetic studies of thymidine phosphorylase from mouse liver.

Initial velocity and product inhibition studies of thymidine phosphorylase from mouse liver revealed that the basic reaction mechanism of this enzyme is a rapid equilibrium random bi-bi mechanism with an enzyme-phosphate-thymine dead-end complex. Thymine displayed both substrate inhibition and nonlinear product inhibition, i.e., slope and intercept replots vs. 1/[thymine] were nonlinear, indicating that there is more than one binding site on the enzyme for thymine and that when thymine is bound to one of these sites, the enzyme is inhibited. Furthermore, both thymidine and phosphate showed "cooperative effects" in the presence of thymine at concentrations above 60 microM, suggesting that the enzyme may have multiple interacting allosteric and/or catalytic sites. The deoxyribosyl transferase reaction catalyzed by this enzyme is phosphate-dependent, requires nonstoichiometric amounts of phosphate, and can proceed by an "enzyme-bound" 2-deoxyribose 1-phosphate intermediate. These findings are in accord with the rapid equilibrium random bi-bi mechanism and demonstrate that deoxyribosyl transfer by this enzyme involves an indirect-transfer mechanism. These results strongly suggest that phosphorolysis and deoxyribosyl transfer are catalyzed by the same site on thymidine phosphorylase.

N-acetylglutamate synthetase (NAGS) deficiency: diagnosis, clinical observations and treatment.

NAGS plays an important role in the short term regulation of urea synthesis. This enzyme has extensively been studied by Shigesada and Tatibana in rodents 1 ,2,3. We have purified human NAGS in order to find out what substrate concentrations should be used in an assay for human liver. The kinetic results including product inhibition studies are compatible with a rapid equilibrium random bibi mechanism. In the absence of arginine the Km are for Acetyl CoA 4.4 mmol/1, for glutamate 8.2 mmol/1. The Ki of N-acetylglutamate is at 0.5 mmol/1. Arginine approximately triples the activity (half maximum activation at about 30 µmol/1), and appears to lower the Km of acetyl CoA, but not of glutamate4.

Steady state kinetics of dihydropteridine reductase: Initial velocity and inhibition studies

We report results of steady state kinetic studies of dihydropteridine reductase from bovine liver. Initial velocity analysis excluded the possibility of a rapid equilibrium ordered mechanism, but could not decisively differentiate between a ping-pong mechanism and a sequential mechanism. Product inhibition patterns with NAD + were competitive against NADH and noncompetitive against quinonoid dihydropterin, thus excluding a ping-pong mechanism and a rapid equilibrium random bi-bi mechanism. The possibility that a dead-end complex is formed between enzyme, pterin, and NAD + in this last mechanism was excluded by the observation that the dead-end inhibitor, aminopterin, is uncompetitive against NADH and noncompetitive against the pterin. Another dead-end inhibitor, Cibacron blue 3G-A, was found to be competitive against NADH and noncompetitive against the pterin. We conclude that the kinetic mechanism of dihydropteridine reductase is ordered bi-bi with the binding of NADH first and the release of NAD + last.

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.

Evidence for cooperative effects in the exchange reaction catalysed by the oxoglutarate translocator of rat-heart mitochondria.

The initial rates of the exchange external oxoglutarate/internal malate through the inner membrane of rat-heart mitochondria, for various concentrations of the two substrates, have been reinvestigated for an extended range of concentrations of the external oxoglutarate. This has been made possible by use of the inhibitor-stop technique that allows 100 times smaller incubation times than the centrifugation-stop technique used previously. Under the experimental conditions the uptake of the external-labelled oxoglutarate into the mitochondrial-matrix space is mediated by the oxoglutarate translocator performing a ono-to-one exchange of the anions oxoglutarate (external) and malate (internal). Two intermediary-plateau regions are observed in the kinetic saturation curve of the translocator by the external oxoglutarate, revealing a complex rate equation which is found to be the product of two one-substrate functions. Analysing these features it is shown that the model, proposed earlier, of a "double carrier" as catalyst in a rapid-equilibrium random bi-bi mechanism, is still applicable but that several external binding sites have to be considered. As already noticed the external and the internal substrates bind to their respective sites independently of each other. Furthermore, some additional requirements imposed by the observed kinetics suggest that the exchange reaction is performed by only one translocator species made of identical interacting subunits. The anion exchange is tentatively viewed as a rotation of a subunit around an axis situated in the plane of the membrane after two independent local configuration changes induced by the binding of the two substrates on this subunit.

Pigeon liver diacetyl reductase kinetic and thermodynamic studies with NADH as coenzyme

1. 1.The dependence of the initial rate of the reaction catalyzed by pigeon liver diacetyl reductase on the concentration of substrates (diacetyl and NADH) and the product (acetoin and NAD +) inhibition pattern are consistent with both a rapid equilibrium random Bi-Bi mechanism with two dead-end ternary complexes and a Theorell-Chance one. 2. 2.The inhibition patterns with acetone and pentane-3-one (competitive for diacetyl and uncompetitive for NADH) discard the random Bi-Bi mechanism and prove that the reaction follows that of Theorell-Chance with NADH as the leading substrate. 3. 3. K m diacetyl (3.1 mM), K m NADNl (100 μM), K s diacetyl (5 mM), K s NADH (162 μM), activation energy (13.8 kcal/mole), ΔH* (13.3 kcal/mole), ΔF* (14.5 kcal/mole) and ΔS* (= −4 cal/mole per degree) were estimated.

Kinetic Mechanism of Glycogen Synthase D from Human Polymorphonuclear Leukocytes

The steady state bisubstrate kinetics of the reaction UDP-glucose + glycogen ⇄ UDP + glycogen catalyzed by glycogen synthase D (UDP-glucose:glycogen α-4-glucosyltransferase, EC 2.4.1.11) was studied, using 14C isotope transfer rates. The reaction rates were determined at varying concentrations of either substrate at constant amounts of the other substrate. The inhibition patterns of UDP as well as the activation patterns of the activator glucose 6-phosphate toward either substrate were determined. UDP was found to act competitively when the substrate UDP-glucose was varied, and noncompetitively when glycogen was the varied substrate, while glucose 6-phosphate showed an intersecting pattern toward glycogen and an equilibrium ordered pattern toward UDP-glucose. The reciprocal plots were found to be linear in all cases. On the basis of the results, it is concluded that glycogen synthase D from human polymorphonuclear leukocytes has a rapid equilibrium random bi-bi mechanism, in which the attachment of the activator glucose 6-phosphate is a necessary prerequisite for the addition of the substrate UDP-glucose (and UDP in the reverse reaction).

Kinetic studies of beef liver diacetyl reductase

1. 1. The dependence of the beef liver diacetyl reductase (acetoin:NAD oxido-reductase, EC 1.1.1.5) reaction rate on the concentration of substrates and the product inhibition pattern are investigated at optimum pH. The Michaelis and dissociation constants for the reaction in the direction diacetyl→acetoin are evaluated. 2. 2. The results are consistent with the features of both a Theorell-Chance and a rapid equilibrium random Bi-Bi mechanism with two dead-end ternary complexes. However, it is concluded that the Theorell-Chance mechanism is the more probable one. 3. 3. Km values for diacetyl and NADH seem to indicate that the enzyme is operative under normal physiological conditions.

Kinetic Mechanism of Rabbit Muscle Glycogen Phosphorylase a

Abstract Isotope-exchange rates at chemical equilibrium were determined for the glycogen-α-d-glucopyranose 1-phosphate-Pi system in the presence of phosphorylase a. Exchange of 32P from α-d-glycopyranose 1-phosphate (glucose-1-P) into Pi and exchange of 14C from glucose-1-P into glycogen were followed simultaneously by the use of glucose-1-P containing both isotopes. Concentrations of glucose-1-P and Pi were varied together in their equilibrium ratio at constant glycogen concentration, and the concentration of glycogen was varied at fixed concentrations of phosphates. Exchange rates for the two isotopes were equal under all conditions (with the possible exception of measurements at the highest concentrations of the phosphates) and gave linear reciprocal plots. One exception was noted in which the reciprocal plot was concave upward at low concentrations of substrate, probably because of allosteric effects. The results support a rapid equilibrium mechanism. Initial velocity of the reaction in the absence of product was determined by the use of an isotopic assay. The results were characteristic of a sequential mechanism. Isotope-exchange rates were also determined under nonequilibrium conditions. Exchange of 32P from glucose-1-P into Pi was followed as a function of glycogen concentration at a fixed concentration of glucose-1-P and several fixed concentrations of Pi. The exchange was also followed as a function of glucose-1-P concentration at fixed glycogen concentration and several fixed Pi concentrations. Similar experiments were done while following the exchange from Pi into glucose-1-P. These experiments are equivalent to conventional product inhibition experiments. The results indicate that the two phosphates are noncompetitive inhibitors with respect to glycogen, but are competitive with respect to one another. Our conclusion is that phosphorylase a has a rapid equilibrium Random Bi-Bi mechanism involving binary complexes of enzyme with glycogen, glucose-1-P, and Pi, and ternary complexes of enzyme with glycogen and glucose-1-P and with glycogen and Pi.