Gamma-glutamyltransferase: Substrate inhibition, kinetic mechanism, and assay conditions.

1976 ◽  
Vol 22 (4) ◽  
pp. 417-421 ◽  
Author(s):  
J H Stromme ◽  
L Theodorsen
Keyword(s):  
Clinical Chemistry ◽  
Substrate Inhibition ◽  
Kinetic Mechanism ◽  
Reaction Conditions ◽  
Gamma Glutamyltransferase ◽  
Gamma Glutamyl ◽  
Dead End ◽  
Ping Pong ◽  
Competitive Substrate ◽  
Assay Conditions

Abstract Gamma-glutamyltransferase activity in serum is shown to be competitively inhibited by the two substrates gamma-glutamyl-4-nitroanilide and glycylglycine. Awareness of this is of importance when one is choosing final reaction conditions for the assay of the enzyme. Gamma-glutamyltransferase probably acts by a "ping-pong bi-bi" kinetic mechanism, which fits with the double competitive substrate inhibition demonstrated. The product, 4-nitro-aniline, appears to be an uncompetitive dead-end inhibitor of both substrates. Various amino acids, particularly glycine and L-alanine, inhibit the enzyme. Their inhibition patterns are uncompetitive with glycylglycine and competitive with gamma-glutamyl-4-nitroanilide. On the basis of the present and other studies, the Scandinavian Society for Clinical Chemistry and Clinical Physiology is going to recommend for routine use a gamma-glutamyltransferase method in which the final concentrations of gamma-glutamyl-4-nitroanilide and glycylglycine are 4 and 75 mmol/liter, respectively.

Gamma-Glutamyltransferase: kinetic properties and assay conditions when gamma-glutamyl-4-nitroanilide and its 3-carboxy derivative are used as donor substrates.

1977 ◽  
Vol 23 (1) ◽  
pp. 79-85 ◽  
Author(s):  
L M Shaw ◽  
J W London ◽  
D Fetterolf ◽  
D Garfinkel
Keyword(s):  
Enzyme Mechanism ◽  
Simultaneous Occurrence ◽  
Kinetic Properties ◽  
Gamma Glutamyltransferase ◽  
Gamma Glutamyl ◽  
Ping Pong ◽  
Clinical Interest ◽  
Kinetics Of ◽  
Substrate Concentrations ◽  
Assay Conditions

Abstract The kinetics of human serum gamma-glutamyltransferase (EC 2.3.2.2) were investigated, with use of glycylglycine as a gamma-glutamyl acceptor substrate and gamma-glutamyl-4-nitroanilide and its carboxy derivative, gamma-glutamyl-3-carboxy-4-nitroanilide, as donor substrates. The simultaneous occurrence of both gamma-glutamyltransfer and autotransfer was established by descending paper chromatography. Constant-ratio double-reciprocal plots confirm that the enzyme mechanism is nonsequential (ping-pong bi-bi). Inhibition by either donor was not found, and inhibition by glycylglycine was only observed at concentrations above those of clinical interest. Kinetic constants obtained by nonlinear regression analysis of initial velocity data were used to determine reagent substrate concentrations for the assay of this enzyme. An assay with use of 4 mmol of gamma-glutamyl-3-carboxy-4-nitroanilide and 100 mmol of glycylglycine per liter yielded equivalent activities to those by assay with use of 4 mmol of gamma-glutamyl-4-nitroanilide and 40 mmol of glycylglycine per liter. These concentrations of the carboxy donor and glycylglycine are also "cost optimal" and present no procedural problems when used.

scholarly journals The kinetic mechanism and properties of the cytoplasmic acetoacetyl-coenzyme A thiolase from rat liver

1974 ◽  
Vol 139 (1) ◽  
pp. 109-121 ◽  
Author(s):  
B. Middleton
Keyword(s):  
Rat Liver ◽  
Substrate Inhibition ◽  
Kinetic Mechanism ◽  
Competitive Inhibitor ◽  
Acetyl Coa ◽  
Keto Form ◽  
Ping Pong ◽  
Substrate Inhibitor ◽  
The Hill ◽  
Covalent Intermediate

1. Cytoplasmic acetoacetyl-CoA thiolase was highly purified in good yield from rat liver extracts. 2. Mg2+ inhibits the rate of acetoacetyl-CoA thiolysis but not the rate of synthesis of acetoacetyl-CoA. Measurement of the velocity of thiolysis at varying Mg2+ but fixed acetoacetyl-CoA concentrations gave evidence that the keto form of acetoacetyl-CoA is the true substrate. 3. Linear reciprocal plots of velocity of acetoacetyl-CoA synthesis against acetyl-CoA concentration in the presence or absence of desulpho-CoA (a competitive inhibitor) indicate that the kinetic mechanism is of the Ping Pong (Cleland, 1963) type involving an acetyl-enzyme covalent intermediate. In the presence of CoA the reciprocal plots are non-linear, becoming second order in acetyl-CoA (the Hill plot shows a slope of 1.7), but here this does not imply co-operative phenomena. 4. In the direction of acetoacetyl-CoA thiolysis CoA is a substrate inhibitor, competing with acetoacetyl-CoA, with a Ki of 67μm. Linear reciprocal plots of initial velocity against concentration of mixtures of acetoacetyl-CoA plus CoA confirmed the Ping Pong mechanism for acetoacetyl-CoA thiolysis. This method of investigation also enabled the determination of all the kinetic constants without complication by substrate inhibition. When saturated with substrate the rate of acetoacetyl-CoA synthesis is 0.055 times the rate of acetoacetyl-CoA thiolysis. 5. Acetoacetyl-CoA thiolase was extremely susceptible to inhibition by an excess of iodoacetamide, but this inhibition was completely abolished after preincubation of the enzyme with a molar excess of acetoacetyl-CoA. This result was in keeping with the existence of an acetyl-enzyme. Acetyl-CoA, in whose presence the overall reaction could proceed, gave poor protection, presumably because of the continuous turnover of acetyl-enzyme in this case. 6. The kinetic mechanism of cytoplasmic thiolase is discussed in terms of its proposed role in steroid biosynthesis.

gamma-Glutamyltransferase in human serum: an analysis of kinetic models.

1981 ◽  
Vol 27 (2) ◽  
pp. 303-307 ◽  
Author(s):  
H E Solberg ◽  
L Theodorsen ◽  
J H Strømme
Keyword(s):  
Kinetic Models ◽  
Competitive Inhibition ◽  
Acceptor Site ◽  
Velocity Measurements ◽  
Gamma Glutamyltransferase ◽  
Donor Substrate ◽  
Ping Pong ◽  
Decreasing Ph ◽  
Best Fit ◽  
Assay Conditions

Abstract The purpose of the study was to elucidate details of the kinetic model for gamma-glutamyltransferase when assayed with gamma-glutamyl-3-carboxy-4-nitroanilide and glycylglycine as substrates. Data from several sets of initial velocity measurements were fitted by nonlinear regression to a set of different kinetic models. gamma-Glutamyltransferase acts by a "ping-pong bi-bi" mechanism. A model encompassing transfer and autotransfer, competitive inhibition by the acceptor substrate, and no inhibition by the donor substrate gives the best fit to the experimental data. Effects of spectrophotometric nonlinearity may simulate noncompetitive inhibition by the donor substrate. The nonlinearity is dependent on the absorption of the incubation mixture and therefore is related to the concentration of the donor substrate and the wavelength (405-412 nm) used to monitor the reaction. With decreasing pH the autotransfer fraction decreases and the binding of the donor substrate to the acceptor site increases, simulating an increased competitive inhibition by the donor substrate. These results are of importance when elaborating optimum assay conditions for gamma-glutamyltransferase in serum.

scholarly journals Purification and steady-state kinetics of adenosine 5′-pyrophosphate sulphurylase from baker's yeast

1977 ◽  
Vol 165 (1) ◽  
pp. 149-155 ◽  
Author(s):  
R G Nicholls
Keyword(s):  
Exchange Reaction ◽  
Initial Velocity ◽  
Isotope Exchange ◽  
Substrate Inhibition ◽  
Deae Cellulose ◽  
Product Inhibition ◽  
Steady State Kinetics ◽  
Ping Pong ◽  
Competitive Substrate ◽  
Kinetics Of

ADP sulphurylase (EC 2.7.7.5) was purified by chromatography on Sephadex G-200 and DEAE-cellulose. The enzyme was assayed by measuring the incorporation of [32P]Pi into ADP in the presence of the substrate for the reverse reaction, adenosine 5′-sulphatophosphate. In the concentration ranges investigated, by using initial-velocity, product-inhibition and isotope-exchange studies, the data were consistent with a Ping Pong reaction mechanism, with Km for adenosine 5′-sulphatophosphate of 1.20 +/- 0.08 mM and a Km for Pi of 4.95 +/- 0.15 mM. Competitive substrate inhibition by Pi (Ki = 11.7 +/- 0.3 mM) was found. ADP sulphurylase catalyses a sulphate-independent Pi-ADP exchange reaction, the kinetics of which are consistent with the kinetics of the overall reaction, inconsistent with the assay of Burnell & Anderson [(1973) Biochem. J. 133, 417-428], which is based on a sulphate-dependent Pi-ADP exchange reaction.

Mechanism of a long-chain fatty aldehyde dehydrogenase induced during the development of bioluminescence in Beneckea harveyi

1983 ◽  
Vol 61 (5) ◽  
pp. 301-306 ◽  
Author(s):  
Andrew Bognar ◽  
Edward Meighen
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Substrate Inhibition ◽  
Kinetic Mechanism ◽  
Fatty Aldehyde ◽  
Rate Limiting Step ◽  
Dead End ◽  
Sequential Mechanism ◽  
High Concentrations ◽  
Substrate Protection ◽  
Long Chain

The kinetic mechanism of a long-chain aldehyde dehydrogenase that is induced during the development of bioluminescence in Beneckea harveyi has been investigated. Parallel lines were obtained in Lineweaver–Burk plots with NAD+ and long-chain aldehydes (heptaldehyde, nonylaldehyde). However, product and dead-end inhibitor studies, substrate protection (NAD+, aldehyde) against inactivation with N-ethylmaleimide, and in particular, a noncompetitive substrate inhibition pattern with aldehyde at high concentrations showed that aldehyde dehydrogenase had a sequential mechanism. The data were consistent with a nonrapid equilibrium random mechanism with a preferred order of addition of substrates (NAD+, aldehyde) and an ordered release of products with NADH release being the last and rate-limiting step in the reaction, a mechanism very similar to that found for short-chain mammalian aldehyde dehydrogenases. The present experiments emphasize the caution that must be taken in interpreting parallel patterns in initial velocity experiments, as well as the difficulty in classifying sequential enzyme mechanisms as either strictly ordered or random.

scholarly journals A steady-state kinetic analysis of the fructose 1,6-bisphosphate-activated pyruvate kinase from Carcinus maenas hepatopancreas

1980 ◽  
Vol 185 (2) ◽  
pp. 289-299 ◽  
Author(s):  
I G Giles ◽  
P C Poat
Keyword(s):  
Pyruvate Kinase ◽  
Substrate Inhibition ◽  
Carcinus Maenas ◽  
Product Inhibition ◽  
Dead End ◽  
Steady State Kinetic ◽  
Competitive Substrate ◽  
Fructose 1,6 Bisphosphate ◽  
Inhibition Studies ◽  
State Kinetic

1. An investigation of the reaction mechanism of the fructose 1,6-bisphosphate-activated pyruvate kinase isolated from the hepatopancreas of the crab Carcinus maenas was conducted. The enzyme was assayed in the presence of 500 microns-fructose 1,6-bisphosphate, 75 mM-KCl and 8 mM-Mg2+free at 25 degrees C. The results are consistent with a rapid-equilibrium random mechanism. 2. Evidence is presented that suggests the formation of two mixed-substrate-product dead-end complexes, enzyme-ADP-pyruvate and enzyme-ADP-ATP. 3. Competitive substrate inhibition was observed for both substrates, ADP and phosphoenolpyruvate, suggesting the formation of the complexes enzyme-ADP-ADP and enzyme-phosphoenolpyruvate-phosphoenolpyruvate in the suggested mechanism. 4. Data from the ATP product-inhibition studies indicate the formation of the complex enzyme-ATP-ATP. This suggests that in the reverse reaction ATP also will show substrate inhibition. 5. The presence of a saturating concentration of fructose 1,6-bisphosphate does not cause full activation of the purified preparations of the enzyme. 6. Pyruvate kinase activity in the supernatant of a hepatopancreas homogenate was completely activated by fructose 1,6-bisphosphate, suggesting that the binding of this ligand to the purified pyruvate kinase was impaired.

.epsilon.-(.gamma.-Glutamyl)lysine: Hydrolysis by .gamma.-Glutamyltransferase of Different Origins, When Free or Protein Bound

1995 ◽  
Vol 43 (8) ◽  
pp. 1977-1981 ◽  
Author(s):  
Katsuya Seguro ◽  
Yoshiyuki Kumazawa ◽  
Tomoko Ohtsuka ◽  
Hiroyuki Ide ◽  
Noriki Nio ◽  
...  
Keyword(s):  
Gamma Glutamyltransferase ◽  
Gamma Glutamyl ◽  
Different Origins

scholarly journals The kinetic mechanism of 3-hydroxy-3-methylglutaryl-coenzyme A synthase from baker's yeast

1972 ◽  
Vol 126 (1) ◽  
pp. 35-47 ◽  
Author(s):  
B. Middleton
Keyword(s):  
Ph Value ◽  
Acyl Chain ◽  
Substrate Inhibition ◽  
Kinetic Mechanism ◽  
Hydroxyl Group ◽  
Acetyl Coa ◽  
Hydrophobic Region ◽  
Active Centre ◽  
Acyl Chain Length ◽  
Inhibition Pattern

1. The effect of independent variation of both acetyl-CoA and acetoacetyl-CoA on the initial velocity at pH8.0 and pH8.9 gives results compatible with a sequential mechanism involving a modified enzyme tentatively identified as an acetyl-enzyme, resulting from the reaction with acetyl-CoA in the first step of a Ping Pong (Cleland, 1963a) reaction. 2. Acetoacetyl-CoA gives marked substrate inhibition that is competitive with acetyl-CoA. This suggests formation of a dead-end complex with the unacetylated enzyme and is in accord with the inhibition pattern given by 3-oxohexanoyl-CoA, an inactive analogue of acetoacetyl-CoA. 3. The inhibition pattern given by products of the reaction is compatible with the above mechanism. CoA gives mixed inhibition with respect to both substrates, whereas dl-3-hydroxy-3-methylglutaryl-CoA competes with acetyl-CoA but gives uncompetitive inhibition with respect to acetoacetyl-CoA. 4. 3-Hydroxy-3-methylglutaryl-CoA analogues lacking the 3-hydroxyl group are found to compete, like 3-hydroxy-3-methylglutaryl-CoA, with acetyl-CoA but have Ki values ninefold higher, indicating the importance of the 3-hydroxyl group in the interaction. 5. A comparison of inhibition by CoA and desulpho-CoA at pH8.0 and pH8.9 shows that at the higher pH value a kinetically significant reversal of the formation of acetyl-enzyme can occur. 6. Acetyl-CoA homologues do not act as substrates and compete only with acetyl-CoA. A study of the variation of Ki with acyl-chain length suggests the presence near the active centre of a hydrophobic region. 7. These results are discussed in terms of a kinetic mechanism in which there is only one CoA-binding site the specificity of which is altered by acetylation of the enzyme. 8. The rate of 3-hydroxy-3-methylglutaryl-CoA synthesis in yeast is calculated from the kinetic constants determined for purified 3-hydroxy-3-methylglutaryl-CoA synthase and from estimates of the physiological substrate concentrations. The rate of synthesis of 12nmol of 3-hydroxy-3-methylglutaryl-CoA/min per g wet wt. of yeast is still greater than the rate of utilization in spite of the extremely low (calculated) acetoacetyl-CoA concentration (1.8nm).

scholarly journals Steady-state kinetic mechanism of the NADP+- and NAD+-dependent reactions catalysed by betaine aldehyde dehydrogenase from Pseudomonas aeruginosa

2000 ◽  
Vol 352 (3) ◽  
pp. 675-683 ◽  
Author(s):  
Roberto VELASCO-GARCÍA ◽  
Lilian GONZÁLEZ-SEGURA ◽  
Rosario A. MUÑOZ-CLARES
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Steady State ◽  
Aldehyde Dehydrogenase ◽  
Substrate Inhibition ◽  
Kinetic Mechanism ◽  
Betaine Aldehyde Dehydrogenase ◽  
Betaine Aldehyde ◽  
Metabolic Role ◽  
Steady State Kinetic ◽  
State Kinetic

Betaine aldehyde dehydrogenase (BADH) catalyses the irreversible oxidation of betaine aldehyde to glycine betaine with the concomitant reduction of NAD(P)+ to NADP(H). In Pseudomonas aeruginosa this reaction is a compulsory step in the assimilation of carbon and nitrogen when bacteria are growing in choline or choline precursors. The kinetic mechanisms of the NAD+- and NADP+-dependent reactions were examined by steady-state kinetic methods and by dinucleotide binding experiments. The double-reciprocal patterns obtained for initial velocity with NAD(P)+ and for product and dead-end inhibition establish that both mechanisms are steady-state random. However, quantitative analysis of the inhibitions, and comparison with binding data, suggest a preferred route of addition of substrates and release of products in which NAD(P)+ binds first and NAD(P)H leaves last, particularly in the NADP+-dependent reaction. Abortive binding of the dinucleotides, or their analogue ADP, in the betaine aldehyde site was inferred from total substrate inhibition by the dinucleotides, and parabolic inhibition by NADH and ADP. A weak partial uncompetitive substrate inhibition by the aldehyde was observed only in the NADP+-dependent reaction. The kinetics of P. aeruginosa BADH is very similar to that of glucose-6-phosphate dehydrogenase, suggesting that both enzymes fulfil a similar amphibolic metabolic role when the bacteria grow in choline and when they grow in glucose.

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