Kinetic mechanism of phosphorylase a. I. Initial velocity studies

Initial rate studies on rabbit muscle phosphorylase a were carried out in order to assign a kinetic mechanism to this enzyme, which plays an important role in the control of glycogen metabolism. Initial velocities were measured with varied concentrations of both substrates in each reaction direction, both in the presence and the absence of the modifier AMP. Data were analyzed with double-reciprocal plots and secondary replots of slopes and intercepts to yield kinetically derived dissociation constants which may be compared with dissociation constants determined by independent methods. Inhibition studies using UDP-glucose as a substrate analogue are also reported. Inhibition is competitive with glucose 1-phosphate and P1 and noncompetitive with glycogen.A suitable rate equation for this system has been derived, and it should apply to other two-substrate enzyme–modifier systems exhibiting similar kinetics. The results of this detailed kinetic analysis, in conjunction with the isotope exchange studies at equilibrium which are reported in the following paper, suggest the kinetic mechanism of phosphorylase a to be rapid equilibrium random Bi Bi, i.e. random addition of substrates to the enzyme, with ternary complex interconversion as the rate-limiting step in the reaction sequence.

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Kinetic mechanism of phosphorylase a, II. Isotope exchange studies at equilibrium

Canadian Journal of Biochemistry ◽

10.1139/o70-118 ◽

1970 ◽

Vol 48 (7) ◽

pp. 755-758 ◽

Cited By ~ 18

Author(s):

H. D. Engers ◽

W. A. Bridger ◽

N. B. Madsen

Keyword(s):

Exchange Rates ◽

Isotope Exchange ◽

Initial Rate ◽

Kinetic Mechanism ◽

Rabbit Muscle ◽

Glucose Inhibition ◽

Rate Limiting Step ◽

Equilibrium Exchange ◽

Muscle Phosphorylase ◽

Rate Limiting

In order to confirm the kinetic mechanism which was proposed for rabbit muscle phosphorylase a on the basis of initial rate studies and UDP-glucose inhibition experiments, isotope exchange studies at equilibrium were performed, both in the presence and absence of the modifier AMP.Both the 14C-glucose [Formula: see text] and the [Formula: see text]1-phosphate equilibrium exchange rates increased to a maximum as the concentrations of the varied substrates became saturating, either in the presence or absence of AMP. The plateaus observed in these experiments indicate the lack of inhibition of the exchange of one pair of substrates when the concentration of the other substrate pair was raised, and confirms the proposed random addition of substrates to the enzyme.The fact that similar exchange rates were observed for either reaction direction reinforced the concept that rapid equilibrium conditions apply to the phosphorylase a mechanism; i.e. the interconversion of the ternary complexes tends to be the rate-limiting step in the reaction sequence.Maximal velocities determined from initial rate data reported in the previous paper agreed with those calculated from isotope exchange rates.

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Human glutamic-γ-semialdehyde dehydrogenase. Kinetic mechanism

Biochemical Journal ◽

10.1042/bj2610935 ◽

1989 ◽

Vol 261 (3) ◽

pp. 935-943 ◽

Cited By ~ 11

Author(s):

C Forte-McRobbie ◽

R Pietruszko

Keyword(s):

Kinetic Mechanism ◽

Product Inhibition ◽

Maximal Velocity ◽

Semialdehyde Dehydrogenase ◽

Rate Limiting Step ◽

Dead End ◽

Inhibition Studies ◽

Rate Limiting ◽

Competitive Pattern ◽

Pattern Versus

The kinetic mechanism of homogeneous human glutamic-gamma-semialdehyde dehydrogenase (EC 1.5.1.12) with glutamic gamma-semialdehyde as substrate was determined by initial-velocity, product-inhibition and dead-end-inhibition studies to be compulsory ordered with rapid interconversion of the ternary complexes (Theorell-Chance). Product-inhibition studies with NADH gave a competitive pattern versus varied NAD+ concentrations and a non-competitive pattern versus varied glutamic gamma-semialdehyde concentrations, whereas those with glutamate gave a competitive pattern versus varied glutamic gamma-semialdehyde concentrations and a non-competitive pattern versus varied NAD+ concentrations. The order of substrate binding and release was determined by dead-end-inhibition studies with ADP-ribose and L-proline as the inhibitors and shown to be: NAD+ binds to the enzyme first, followed by glutamic gamma-semialdehyde, with glutamic acid being released before NADH. The Kia and Kib values were 15 +/- 7 microM and 12.5 microM respectively, and the Ka and Kb values were 374 +/- 40 microM and 316 +/- 36 microM respectively; the maximal velocity V was 70 +/- 5 mumol of NADH/min per mg of enzyme. Both NADH and glutamate were product inhibitors, with Ki values of 63 microM and 15,200 microM respectively. NADH release from the enzyme may be the rate-limiting step for the overall reaction.

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Substrate specificity of sheep liver sorbitol dehydrogenase

Biochemical Journal ◽

10.1042/bj3300479 ◽

1998 ◽

Vol 330 (1) ◽

pp. 479-487 ◽

Cited By ~ 28

Author(s):

I. Rune LINDSTAD ◽

Peter KÖLL ◽

John S. McKINLEY-McKEE

Keyword(s):

Substrate Specificity ◽

Ternary Complex ◽

Kinetic Mechanism ◽

Sorbitol Dehydrogenase ◽

Hydroxyl Group ◽

Enzyme Active Site ◽

Sheep Liver ◽

Rate Limiting Step ◽

Steady State Kinetics ◽

Rate Limiting

The substrate specificity of sheep liver sorbitol dehydrogenase has been studied by steady-state kinetics over the range pH 7-10. Sorbitol dehydrogenase stereo-selectively catalyses the reversible NAD-linked oxidation of various polyols and other secondary alcohols into their corresponding ketones. The kinetic constants are given for various novel polyol substrates, including L-glucitol, L-mannitol, L-altritol, D-altritol, D-iditol and eight heptitols, as well as for many aliphatic and aromatic alcohols. The maximum velocities (kcat) and the substrate specificity-constants (kcat/Km) are positively correlated with increasing pH. The enzyme-catalysed reactions occur by a compulsory ordered kinetic mechanism with the coenzyme as the first, or leading, substrate. With many substrates, the rate-limiting step for the overall reaction is the enzyme-NADH product dissociation. However, with several substrates there is a transition to a mechanism with partial rate-limitation at the ternary complex level, especially at low pH. The kinetic data enable the elucidation of new empirical rules for the substrate specificity of sorbitol dehydrogenase. The specificity-constants for polyol oxidation vary as a function of substrate configuration with D-xylo > d-ribo > L-xylo > d-lyxo ≈ l-arabino > D-arabino > l-lyxo. Catalytic activity with a polyol or an aromatic substrate and various 1-deoxy derivatives thereof varies with -CH2OH >-CH2NH2 >-CH2OCH3 ≈-CH3. The presence of a hydroxyl group at each of the remaining chiral centres of a polyol, apart from the reactive C2, is also nonessential for productive ternary complex formation and catalysis. A predominantly nonpolar enzymic epitope appears to constitute an important structural determinant for the substrate specificity of sorbitol dehydrogenase. The existence of two distinct substrate binding regions in the enzyme active site, along with that of the catalytic zinc, is suggested to account for the lack of stereospecificity at C2 in some polyols.

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Enhancement of hepatic glycogen by gluconeogenic precursors: substrate flux or metabolic control?

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1990.258.6.e899 ◽

1990 ◽

Vol 258 (6) ◽

pp. E899-E906 ◽

Cited By ~ 4

Author(s):

J. H. Youn ◽

R. N. Bergman

Keyword(s):

Glycogen Metabolism ◽

Hepatic Glycogen ◽

Glycogen Accumulation ◽

Major Mechanism ◽

Rate Limiting Step ◽

Key Enzyme ◽

Glucose Phosphorylation ◽

Rate Limiting ◽

Gluconeogenic Substrates

After a meal or glucose load, most carbons of hepatic glycogen are derived from gluconeogenesis. In vitro, hepatic glycogen accumulation is sluggish with glucose alone but markedly enhanced in the presence of gluconeogenic substrates. These findings conflict with the classical view that glucose is the major precursor of hepatic glycogen and have been termed the "glucose paradox." In this review, we attempt to elucidate the central mechanism underlying the glucose paradox by critically examining the in vitro data of hepatic glycogen accumulation. Our analysis is inconsistent with the current hypothesis that glucose phosphorylation is rate limiting for hepatic glycogen accumulation from glucose and that gluconeogenesis enhances glycogen accumulation primarily by increasing substrate flux to the hepatic glucose 6-phosphate pool. Instead, our analysis leads us to the conclusion that the rate-limiting step is the net incorporation of glucose 6-phosphate into glycogen, which is synergistically facilitated with glucose and gluconeogenic substrates. Thus gluconeogenic substrates are involved in the regulation of key enzyme(s) of glycogen metabolism. In addition, in the livers from fasted rats there is substantial cycling through glycogen, and that suppression of glycogen degradation may be a major mechanism in the enhancement of glycogen accumulation by gluconeogenic substrates. Thus we propose a specific hypothesis of the role of gluconeogenic substrates in glycogen metabolism (i.e., inhibition of phosphorylase), which can be tested by future studies.

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Mechanism of the 2,3-diphosphoglycerate-dependent phosphoglycerate mutase from rabbit muscle

Biochemical Journal ◽

10.1042/bj1300397 ◽

1972 ◽

Vol 130 (2) ◽

pp. 397-410 ◽

Cited By ~ 38

Author(s):

H. G. Britton ◽

J. B. Clarke

Keyword(s):

Chemical Equilibrium ◽

Phosphoglycerate Mutase ◽

Rabbit Muscle ◽

Kinetic Measurements ◽

Rate Limiting Step ◽

Ping Pong ◽

Sequential Mechanism ◽

Rate Limiting ◽

Kinetics Of ◽

Intramolecular Transfer

1. The properties and kinetics of the 2,3-diphosphoglycerate-dependent phosphoglycerate mutases are discussed. There are at least three possible mechanisms for the reaction: (i) a phosphoenzyme (Ping Pong) mechanism; (ii) an intermolecular transfer of phosphate from 2,3-diphosphoglycerate to the substrates (sequential mechanism); (iii) an intramolecular transfer of phosphate. It is concluded that these mechanisms cannot be distinguished by conventional kinetic measurements. 2. The fluxes for the different mechanisms are calculated and it is shown that it should be possible to distinguish between the mechanisms by appropriate induced-transport tests and by comparing the fluxes of 32P- and 14C-labelled substrates at chemical equilibrium. 3. With 14C-labelled substrates no induced transport was found over a wide concentration range, and with 32P-labelled substrates co-transport occurred that was independent of concentration over a twofold range. 14C-labelled substrates exchange at twice the rate of 32P-labelled substrates at chemical equilibrium. The results were completely in accord with a phosphoenzyme mechanism and indicated a rate constant for the isomerization of the phosphoenzyme of not less than 4×106s−1. The intramolecular transfer of phosphate (and intermolecular transfer between two or more molecules of substrate) were completely excluded. The intermolecular transfer of phosphate from 2,3-diphosphoglycerate would have been compatible with the results only if the Km for 2-phosphoglycerate had been over 7.5-fold smaller than the observed value and if an isomerization of the enzyme-2,3-diphosphoglycerate complex had been the major rate-limiting step in the reaction. 4. The very rapid isomerization of the phosphoenzyme that the experiments demonstrate suggests a mechanism that does not involve a formal isomerization. According to this new scheme the enzyme is closely related mechanistically and perhaps evolutionarily to a 2,3-diphosphoglycerate diphosphatase.

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Reactions of 1,3-diacylthioureas with methoxide ion and with amines

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19870120 ◽

1987 ◽

Vol 52 (1) ◽

pp. 120-131 ◽

Cited By ~ 2

Author(s):

Jaromír Kaválek ◽

Josef Jirman ◽

Vojeslav Štěrba

Keyword(s):

Carbonyl Group ◽

Rate Constants ◽

Dissociation Constants ◽

Equilibrium Constants ◽

Rate Limiting Step ◽

Limiting Step ◽

Order Of Magnitude ◽

Acetyl Group ◽

Methoxide Ion ◽

Rate Limiting

Rate constants of base-catalyzed methanolysis and dissociation constants in methanol have been determined for benzoylthiourea (II), 1,3-diacetylthiourea (III), 1,3-dibenzoylthiourea (IV), and 1-acetyl-3-benzoylthiourea (V). With the diacyl derivatives III and IV, the reaction of methoxide ion with the neutral substrate is accompanied by that of methoxide with the substrate anion (at higher alkoxide concentrations). Above 0.1 mol l-1 CH3O(-), the rate constants are also affected by medium. The rate of the reaction of neutral diacyl derivative is decreased, and that of the reaction of methoxide with the substrate anion is rapidly increased. The dissociation constant of II is higher than that of acetylthiourea (I) by about one order of magnitude, but the attack of methoxide on the carbonyl group of II is about three times slower than that in I. The benzoyl group at the N1 nitrogen exhibits a greater activating influence (in both the rate and the equilibrium constants) on the other NHCOR group than the acetyl group does. With V the ratio of methanolysis rate constants is 9 : 1 in favour of the acetyl group. The reaction of diacetyl derivative III with 1-butanamine has been followed in butanamine buffers. At the lowest butanamine concentrations, the reaction is second order in the amine, and the rate-limiting step is the proton transfer from the intermediate to the second amine molecule. At the highest butanamine concentrations the reaction becomes first order in the amine, and the rate-limiting step changes to the attack of butanamine on the carbonyl group of diacetyl derivative III.

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Kinetics and mechanism of methanolysis of benzoyl derivatives of substituted phenylureas and phenylthioureas

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19842103 ◽

1984 ◽

Vol 49 (9) ◽

pp. 2103-2110 ◽

Cited By ~ 1

Author(s):

Jaromír Kaválek ◽

Said El Bahaie ◽

Vojeslav Štěrba

Keyword(s):

Nitrogen Atom ◽

Steric Hindrance ◽

Rate Constants ◽

Dissociation Constants ◽

Kinetics And Mechanism ◽

Rate Limiting Step ◽

Limiting Step ◽

Order Of Magnitude ◽

Rate Limiting ◽

Derivatives Of

The methanolysis rate constants and dissociation constants have been measured of benzoyl derivatives of substituted phenylureas and phenylthioureas. The dissociation constants of the thio derivatives are higher by 1 order of magnitude and the rate constants are higher by 2 orders of magnitude than the respective values of the oxygen analogues. Logarithms of the rate and dissociation constants have been correlated with the Hammet σ constant; the ρ constant of the methanolysis of the oxygen derivatives is almost 2x higher than that of the thio derivatives, which is explained by a change in the rate-limiting step. Methylation of the phenyl nitrogen atom increases the acidity by almost 2 orders of magnitude. This effect is due obviously to steric hindrance to the conjugation with the adjacent carbonyl or thiocarbonyl group.

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Kinetic studies of formate dehydrogenase

Biochemical Journal ◽

10.1042/bj1200763 ◽

1970 ◽

Vol 120 (4) ◽

pp. 763-769 ◽

Cited By ~ 27

Author(s):

D. Peacock ◽

D. Boulter

Keyword(s):

Carbon Dioxide ◽

Formate Dehydrogenase ◽

Kinetic Studies ◽

Kinetic Mechanism ◽

Product Inhibition ◽

Reverse Reaction ◽

Enzyme Form ◽

Rate Limiting Step ◽

Measurable Rate ◽

Rate Limiting

1. The kinetic mechanism of formate dehydrogenase is a sequential pathway. 2. The binding of the substrates proceeds in an obligatory order, NAD+ binding first, followed by formate. 3. It seems most likely that the interconversion of the central ternary complex is extremely rapid, and that the rate-limiting step is the formation or possible isomerization of the enzyme–coenzyme complexes. 4. The secondary plots of the inhibitions with HCO3− and NO3− are non-linear, which suggests that more than one molecule of each species is able to bind to the same enzyme form. 5. The rate of the reverse reaction with carbon dioxide at pH6.0 is 20 times that with bicarbonate at pH8.0, although no product inhibition could be detected with carbon dioxide. The low rate of the reverse reaction precluded any steady-state analysis as the enzyme concentrations needed to obtain a measurable rate are of the same order as the Km values for NAD+ and NADH.

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