Alternative mechanism for a bimolecular nonsequential enzymic reaction

1969 ◽  
Vol 47 (2) ◽  
pp. 111-115 ◽  
Author(s):  
R. O. Hurst
Keyword(s):  
Reaction Mechanism ◽  
Product Inhibition ◽  
Alternative Mechanism ◽  
Enzymic Reaction ◽  
Dead End ◽  
Ping Pong ◽  
Inhibition Studies ◽  
Different Order ◽  
Inhibition Pattern

An enzymic reaction mechanism characterized as 'di-Uni Iso Ping Pong' which has the same product inhibition pattern as the 'Ping Pong Bi Bi' mechanism but a different order for the release of products is discussed. A basis for differentiating the two mechanisms by dead-end inhibition studies is given.

scholarly journals The mechanism of the reaction between glutathione and 1-menaphthyl sulphate catalysed by a glutathione S-transferase from rat liver

1973 ◽  
Vol 135 (4) ◽  
pp. 797-804 ◽  
Author(s):  
Brian Gillham
Keyword(s):  
Rat Liver ◽  
Gel Filtration ◽  
Product Inhibition ◽  
Glutathione S Transferase ◽  
Michaelis Constant ◽  
Dead End ◽  
Michaelis Constants ◽  
Inhibition Studies ◽  
Inhibition Pattern ◽  
Mechanism Of The Reaction

1. The glutathione S-transferase that catalyses the reaction of 1-menaphthyl (naphth-1-ylmethyl) sulphate with GSH was purified 76-fold from rat liver. 2. The properties of the purified enzyme were studied by gel filtration and isoelectric focusing. 3. The initial-velocity pattern in the absence of products and the product-inhibition pattern have been determined. These are consistent with an Ordered Bi Bi mechanism in which the GSH adds to the enzyme before 1-menaphthyl sulphate and the products are released in the order SO42−followed by S-(1-menaphthyl)glutathione. 4. Dead-end-inhibition studies with p-aminobenzoic acid, which has been shown to be competitive with GSH and non-competitive with 1-menaphthyl sulphate, support the suggestion that an Ordered Bi Bi mechanism is operative. 5. Values were determined for some of the dissociation and Michaelis constants for the reaction of the substrates and products with the enzyme. 6. It appears that S-(1-menaphthyl)glutathione activates the enzyme when the concentration of GSH is saturating and that of 1-menaphthyl sulphate is low (of the order of its Michaelis constant).

Characterization of the reaction mechanism for the XL-I form of bovine liver xenobiotic/medium-chain fatty acid:CoA ligase

2001 ◽  
Vol 357 (1) ◽  
pp. 283-288 ◽  
Author(s):  
Donald A. VESSEY ◽  
Michael KELLEY
Keyword(s):  
Reaction Mechanism ◽  
Bovine Liver ◽  
Liver Mitochondria ◽  
Product Inhibition ◽  
Bivalent Cation ◽  
Medium Chain ◽  
Apparent Homogeneity ◽  
Ping Pong ◽  
Inhibition Studies

The XL-I form of xenobiotic/medium-chain fatty acid:CoA ligase was purified to apparent homogeneity from bovine liver mitochondria and used to determine the reaction mechanism. A tersubstrate kinetic analysis was conducted by varying the concentrations of ATP, benzoate and CoA in turn. Both ATP and benzoate gave parallel double-reciprocal plots against CoA, which indicates a Ping Pong mechanism, with either pyrophosphate or AMP leaving before the binding of CoA. Addition of pyrophosphate to the assays changed the plots from parallel to intersecting; addition of AMP did not. This indicates that pyrophosphate is the product that leaves before binding of CoA. Based on end-product inhibition studies, it was concluded that the reaction follows a Bi Uni Uni Bi Ping Pong mechanism, with ATP binding first, followed in order by benzoate binding, pyrophosphate release, CoA binding, benzoyl-CoA release and AMP release. A similar mechanism was obtained when the ligase was examined with butyrate as substrate. However, butyrate activation was characterized by a much higher affinity for CoA. This is attributed to steric factors resulting from the bulkier nature of the benzoate molecule. Also, with butyrate there is a bivalent cation activation distinct from that associated with binding to ATP. This activation by excess Mg2+ results in non-linear plots of 1/v against 1/[ATP] for butyrate unless the concentrations of Mg2+ and ATP are varied together.

scholarly journals Human glutamic-γ-semialdehyde dehydrogenase. Kinetic mechanism

1989 ◽  
Vol 261 (3) ◽  
pp. 935-943 ◽  
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.

The analysis of dead-end inhibition patterns for multisubstrate systems

1969 ◽  
Vol 47 (2) ◽  
pp. 91-108
Author(s):  
R. O. Hurst
Keyword(s):  
Reaction Mechanism ◽  
Rate Equation ◽  
Rate Constants ◽  
Additive Effect ◽  
Enzyme Form ◽  
Linear Fashion ◽  
Dead End ◽  
Ping Pong ◽  
Full Rate ◽  
Enzyme Species

The additive effect of a dead-end inhibitor combining in linear fashion with more than one of the enzyme species in a reaction mechanism is demonstrated. An equation for the calculation of the inhibitor constants that are required for the multicombinations of inhibitor from those obtained for the inhibitor combining with only one enzyme form is provided. A method of tabulation of the inhibitor constants with respect to the coefficients of the denominator terms of the full rate-equation for the uninhibited reaction is given, that facilitates the analysis of the inhibition patterns for the several inhibitor complexes that may be formed. The usefulness of the method for calculating the rate constants for a 'Ping Pong Bi Bi' mechanism is illustrated.

scholarly journals A steady-state kinetic study of the reaction catalysed by the secondary-amine mono-oxygenase of Pseudomonas aminovorans

1976 ◽  
Vol 157 (1) ◽  
pp. 197-205 ◽  
Author(s):  
D F Brook ◽  
P J Large
Keyword(s):  
Steady State ◽  
Affinity Chromatography ◽  
Secondary Amine ◽  
Gel Filtration ◽  
Product Inhibition ◽  
Steady State Kinetic ◽  
Ping Pong ◽  
Michaelis Constants ◽  
Inhibition Studies ◽  
State Kinetic

1. Secondary-amine mono-oxygenase (proposed EC group 1.14.99.-) was partially purified from trimethylamine-grown Pseudomonas aminovorans by (NH4)2SO4 fractionation, gel filtration, hydrophobic chromatography on 5-aminopentylamino-Sepharose, and affinity chromatography on Sepharose-bound NADH. 2. Some problems in the affinity-chromatography step are discussed. 3. A steady-state kinetic analysis varying substrate, oxygen and electron-donor concentrations was performed, which, over the concentration range studied, gave a series of families of approximately parallel double-reciprocal plots. From secondary and tertiary plots, Michaelis constants of 0.160 mM, 0.086 mM and 0.121 mM were obtained for dimethylamine, NADPH and oxygen respectively. 4. Product-inhibition studies supported the postulated Hexa Uni Ping Pong (triple-transfer) reaction mechanism.

A SIMPLIFIED APPROACH TO THE USE OF DETERMINANTS IN THE CALCULATION OF THE RATE EQUATION FOR A COMPLEX ENZYME SYSTEM

1967 ◽  
Vol 45 (12) ◽  
pp. 2015-2039 ◽  
Author(s):  
R. O. Hurst
Keyword(s):  
Reaction Mechanism ◽  
Rate Equation ◽  
Rate Constants ◽  
Reaction Mechanisms ◽  
Enzyme System ◽  
Inhibition Mechanism ◽  
Simplified Approach ◽  
Enzymic Reaction ◽  
Dead End ◽  
Search For Information

A standardized method of treating the analysis of enzymic reaction mechanisms by means of determinant expressions is given. The fully expanded polynomial expressions for systems of order three, four, and five are presented and their use described. Application of the method to the analysis of the effect of dead-end inhibitors on enzymic reactions is discussed and the inhibitor constants are evaluated in terms of the rate constants involved in the inhibition mechanism. Examples are given to demonstrate the contribution that inhibitor studies may make in the search for information concerning the nature of the enzymic reaction mechanism, in the calculation of the rate constants, and in the estimation of the proportion of the enzyme distributed between the different enzyme forms involved in the reaction.

A matrix transformation method for writing the rate equations for complementary enzymic reactions

1969 ◽  
Vol 47 (6) ◽  
pp. 643-656 ◽  
Author(s):  
R. O. Hurst
Keyword(s):  
Transformation Method ◽  
Product Inhibition ◽  
Kinetic Constants ◽  
Matrix Transformation ◽  
Rate Equations ◽  
Enzymic Reaction ◽  
Enzyme Intermediates ◽  
Inhibition Pattern

A procedure is outlined for writing two rate equations that will yield the same product-inhibition pattern for an enzymic reaction. The definitions of the kinetic constants and the structure of the distribution equations for the enzyme intermediates as well as the order of interaction of the enzyme with the reactants will be different. A list of several mechanisms which can be qualified in this way is included.

Kinetic Studies of Phosphoribosyl-formylglycineamidine Synthetase

1972 ◽  
Vol 50 (5) ◽  
pp. 490-500 ◽  
Author(s):  
Samuel Y. Chu ◽  
J. Frank Henderson
Keyword(s):  
Ammonium Chloride ◽  
Initial Velocity ◽  
Kinetic Studies ◽  
Product Inhibition ◽  
Free Enzyme ◽  
Ping Pong ◽  
Inhibition Constants ◽  
Inhibition Studies

Initial velocity and product inhibition studies of phosphoribosyl-formylglycineamidine synthetase indicate that the reaction involves a fully ping pong mechanism in which glutamine binds to the free enzyme and glutamate is released before the addition of ATP. ADP is released, and phosphoribosyl-formylglycineamide then binds; the liberation of Pi is rapid, and phosphoribosyl-formylglycineamidine is the last product released from the enzyme. The Km values for glutamine, ATP, and phosphoribosyl-formylglycineamide are 1.1 × 10−4 M, 1.5 × 10−3 M, and 1.1 × 10−4 M, respectively. The Km value for ammonium chloride is 7.5 × 10−3 M, and the ratio of Vmax values with ammonium chloride and glutamine is 1/40. The inhibition constants for FGAM and Pi were calculated to be 1.3 × 10−4 M and 6.45 × 10−3 M, respectively.

scholarly journals Cerebral-cortex hexokinases. Elucidation of reaction mechanisms by substrate and dead-end inhibitor kinetic analysis

1971 ◽  
Vol 123 (5) ◽  
pp. 707-715 ◽  
Author(s):  
H. S. Bachelard ◽  
A. G. Clark ◽  
M. F. Thompson
Keyword(s):  
Reaction Mechanism ◽  
Enzymic Activity ◽  
Significant Inhibition ◽  
Kinetic Constants ◽  
Mitochondrial Activity ◽  
Kinetic Properties ◽  
Extraction And Purification ◽  
Dead End ◽  
Ping Pong ◽  
Atp Analogues

1. The substrate kinetic properties of cerebral hexokinases (mitochondrial and cytoplasmic) were studied at limiting concentrations of both glucose and MgATP2−. Primary plots of the enzymic activity gave no evidence of a Ping Pong mechanism in three types of mitochondrial preparation tested (intact and osmotically disrupted mitochondria, and the purified mitochondrial enzyme), nor in the purified cytoplasmic preparation. 2. Secondary plots of intercepts from the primary plots (1/v versus 1/s) versus reciprocal of second substrate of the mitochondrial activity gave kinetic constants which differed from those obtained directly from the plots of 1/v versus 1/s or of s/v versus s, although the ratios of the derived constants were consistent. The kinetic constants obtained with the cytoplasmic enzyme from primary and secondary plots were consistent. 3. Deoxyglucose, as alternative substrate, inhibited cytoplasmic hexokinase by competition with glucose, but did not compete when MgATP2− was the substrate varied. The Ki for deoxyglucose when glucose concentrations were varied was 0.25mm. 4. A range of ATP analogues was tested as potential substrates and inhibitors of hexokinase activity. GTP, ITP, CTP, UTP and βγ-methylene-ATP did not act as substrates, nor did they cause significant inhibition. Deoxy-ATP proved to be almost as effective a substrate as ATP. AMP inhibited but did not act as substrate. 5. N-Acetyl-glucosamine inhibited all preparations competitively when glucose was varied and non-competitively when MgATP2− was varied. AMP inhibition was competitive when MgATP2− was the substrate varied and non-competitive when glucose was varied. 6. The results are interpreted as providing evidence for a random reaction mechanism in all preparations of brain hexokinase, cytoplasmic and mitochondrial. The kinetic properties and reaction mechanism do not change on extraction and purification of the particulate enzyme. 7. The results are discussed in terms of the participation of hexokinase in regulation of cerebral glycolysis.

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