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P F Cook

Publications and source records attributed to P F Cook.

125 records · Page 7Linked to original sources
PubMed1983

Solvent isotope effects on the reaction catalyzed by yeast hexokinase.

The pH dependence of the maximum velocity (V) for the phosphorylation of glucose, the V/Kglucose and the V/KMgATP have been obtained in H2O and 2H2O. In H2O, V decreases below a pK of 5.8, V/Kglucose decreases below a pK of 6.1 and V/KMgATP decreases below a pK of 6.7. In 2H2O, complex behavior is observed for these parameters as a function of pD. The ratios of the parameters in H2O and 2H2O above their respective pK values give solvent deuterium isotope effects of about 1.5-1.7 for all three parameters. When 1,5-anhydromannitol is used as an alternative substrate, an isotope effect different than unity is obtained only for V/K1,5-anhydromannitol which gives a value of about 0.7. Both the complex pH profiles and the relative magnitude of the isotope effects are interpreted in terms of a pH-dependent change in the E X glucose complex.

Catalysis↗
PubMed1982

Kinetic studies to determine the mechanism of regulation of bovine liver glutamate dehydrogenase by nucleotide effectors.

A combination of kinetic and isotope effect studies in the presence and absence of the effectors ADP and GTP was used to elucidate the mechanism of regulation of bovine liver glutamate dehydrogenase. ADP at low concentrations of glutamate competes with TPN for free enzyme. GTP exhibits a similar effect at high concentrations (100 microM and above). When ADP binds at its allosteric site, it increases the off rates of both alpha-ketoglutarate and TPNH from their product complexes. This results in a decrease in V/K for both substrates, an increase in V, and an increase in the deuterium isotope effects for all three parameters so that they are all about 1.3. The rate of release of glutamate from E-TPNH-glutamate is also apparently enhanced since no substrate inhibition by glutamate is observed in the presence of ADP. The effect of GTP is in opposition to that of ADP in that GTP decreases the off rates for both TPN and glutamate from E-TPN-glutamate as well as the off rates for alpha-ketoglutarate and TPNH. This results in an increase in the V/K's for both substrates, a decrease in V, and a decrease in the deuterium isotope effects for all three parameters to a value of 1. Substrate inhibition by glutamate is also eliminated by GTP probably by preventing any significant accumulation of E-TPNH to which glutamate binds as an inhibitor.

Adenosine Diphosphate↗
PubMed1981

Use of isotope effects and pH studies to determine the chemical mechanism of Bacillus subtilis L-alanine dehydrogenase.

Analysis of deuterium isotope effects with L-alanine-d4 and L-serine-d3, and of pH profiles with the same substrates, shows that L-alanine is sticky (that is, reacts to give products 1-7 times as fast as it dissociates) while L-serin is not. The pH profiles show the following: (1) NH3 and monoanionic amino acids are the substrates; (2) a cationic acid group on the enzyme (probably lysine) with a pK of 9.0-9.6 in E-NAD, but a pK well above 10 in E-NADH, must be protonated for activity and good binding of inhibitors and is probably important for maintaining the proper conformation of the enzyme; (3) A cationic acid group on the enzyme (probably histidine) with a pK around 7 in both E-NAD and E-NADA must be unprotonated for oxidation of amino acids but protonated for binding and reaction of pyruvate. This latter group is the acid-base catalyst for the chemical reaction. In E-NAD, it is so positioned that it can hydrogen bond to (and thus when protonated enhance the binding of) a D-hydroxy or a carbonyl group of an inhibitor, but its state of protonation does not affect the binding of L-lactate or propionate. In E-NADH, it is so placed that it can hydrogen bond to both D- and L-hydroxy groups, as well as in carbonyl groups. A chemical mechanism is postulated in which the dehydrogenation of L-alanine by NAD to produce iminopyruvate is followed by attack of water from the same side from which the hydride was removed. The catalytic histidine transfers a proton from the attacking water to the amino group of the resulting carbinolamine and then removes a proton from the hydroxyl group of the carbinolamine as ammonia is eliminated to give pyruvate.

Alanine↗
PubMed1981

Mechanistic deductions from isotope effects in multireactant enzyme mechanisms.

In the enzymatic mechanism with two or more substrates, comparison of the isotope effects on the maximum velocity and on the apparent V/K values when each substrate concentration is varied allows one to deduce the kinetic mechanism and obtain quantitative information on the relative rates at which substrates dissociate from the enzyme, as opposed to undergoing reaction to give products. Theory is also presented for using the effects of other reactants on the apparent isotope effects determined by the equilibrium perturbation method to determine the same information. With liver alochol dehydrogenase, DPN is not released at an appreciable rate from the E-DPN-cyclohexanol complex, while cyclohexanol is released much more rapidly than it reacts to give products, so that the mechanism appears ordered. With DPNH and cyclohexanone, however, the reaction is random since DPNH can be released from the ternary complex at a finite rate. With yeast alcohol dehydrogenase, acetone, when present, prevents DPNH release from the enzyme so that the mechanism at equal rates from E-DPN-2-propanol so tht the reaction is random in this direction.

Alcohol Oxidoreductases↗
PubMed1981

pH variation of isotope effects in enzyme-catalyzed reactions. 2. Isotope-dependent step not pH dependent. Kinetic mechanism of alcohol dehydrogenase.

Theory is developed for th pH dependence of isotope effects in a mechanism where a pH-dependent step precedes the isotope-sensitive bond-breaking step, and the rate of the latter varies only slightly with the state of protonation of the acid-base catalytic group on the enzyme. In such a mechanism, the isotope effects fall to 1.0 in the forward direction and to the equilibrium isotope effect in the reverse direction at pH values where the pH-sensitive step becomes totally rate limiting in the reverse direction. This model accurately describes the kinetics of yeast alcohol dehydrogenase, where V/Kacetone and the isotope effects on V2-propanol and V/K2-propanol decrease above a pK of 8.8 (both isotope effects becoming 1.0 at pH 10). The model also fits the kinetics of liver alcohol dehydrogenase, where Vcyclohexanol and V/Kcyclohexanol decrease below pKs of 6.2 and 7.1, and above pKs of 9.5 and 10.3. pKi trifluoroethanol decreases below a pK of 7.2, and above pK of 10.1, while pKi isobutyramide drops above a pK of 10.0. Vcyclohexanone decreases above a pK of 8.4 while V/Kcyclohexanone decreases above pKs of 8.8 and 9.7. Isotope effects on V/Kcyclohexanol and V/Kcyclohexanone decrease above identical pKs of 9.4 to values of 1 and 0.88, respectively, at pH 11. Comparison of a value of 2.5 for D(V/Kcyclohexanol) with an average value of 5.53 for T(V/Kcyclohexanol) allowed circulation of 6.3 as the intrinsic deuterium isotope effect. These data suggest that E-DPN-alcohol undergoes a proton transfer to the enzyme to give an EH-DPN-alkoxide complex which can lose its proton at high pH to give E-DPN-alkoxide and that both of these alkoxide complexes undergo hydride transfer to give DPNH and ketone. the alkoxide intermediate is not free to dissociate until it is protonated, either because it is coordinated to Zn or because the enzyme is in a closed catalytic configuration.

Alcohol Oxidoreductases↗
PubMed1980

Primary and secondary deuterium isotope effects on equilibrium constants for enzyme-catalyzed reactions.

Primary deuterium equilibrium isotope effects for the reaction of five secondary alcohols with nicotinamide adenine dinucleotide (DPN) to give reduced deuterionicotinamide adenine dinucleotide (DPND) (cyclohexanol-1-d, 1.18; 2-propanol-2-d, 1.175; threo-DL-isocitrate-2-d, 1.168; L-malate-2-d, 1.173; L-lactate-2-d, 1.19) are all approximately 1.18, while for a primary alcohol, ethanol, the value is 1.07, for an amino acid, L-glutamate-2-d, it is 1.14, and for a hemiacetal, glucose-1-d, it is 1.28. In each case deuterium becomes enriched in the alcohol, amino acid, or hemiacetal with respect to DPNH (TPNH). beta-Secondary equilibrium isotope effects for reduction of ketones by DPNH (cyclohexanone-2,2,6,6-d4, 0.82; acetone-d6, 0.78; pyruvate-d3, 0.83; alpha-ketoglutarate-3,3-d2 reduced to glutamate, 0.898; oxaloacetate-3,3-d2, 0.877; oxaloacetate-3R-d, 0.945) give an average value of 0.946/D, with deuterium becoming enriched in the alcohol or amino acid with respect to the ketone. For reduction of acetaldehyde-1-d by DPNH, the observed value of 0.953 includes the equilibrium effect on the hydration equilibrium in addition to that on the reduction, and the calculated values for reduction of the free aldehyde and the hydrate are 0.78 and 1.07. For reduction of benzaldehyde-1-d, which is not hydrated, the observed value was 0.79. The secondary equilibrium isotope effect for conversion of DPN-4-d to DPNH is 0.89, with deuterium becoming enriched in DPNH, and, for conversion of fumarate-2,3-d2 to malate, the value is 0.69, with deuterium becoming enriched in L-malate. The equilibrium isotope effect for reaction of cyclohexanol-1-d with DPN is temperature independent over the range 15-35 degrees C.

Alcohol Oxidoreductases↗
PubMed1977

Overall mechanism and rate equation for O-acetylserine sulfhydrylase.

Saturation curves for O-acetylserine sulfhydrylase using the substrate analogues O-propionyl-L-serine, O-butyryl-L-serine, and beta-chloro-L-alanine all exhibit substrate inhibition and yield Km values comparable to O-acetyl-L-serine, except the O-butyryl derivative which has a Km 5-fold higher. Since all analogues are used as substrates and yield similar kinetic parameters in most cases, it is possible that they share a common intermediate. This evidence also suggests that specificity of O-acetylserine sulfhydrylase resides in the fact that the beta-substituted moiety on L-serine is a good leaving group. The overall rate equation for O-acetylserine sulfhydrylase was derived. A comparison of the numerical integration of the rate equation and an experimental time course is given.

Alanine↗
PubMed1976

A reaction mechanism from steady state kinetic studies for O-acetylserine sulfhydrylase from Salmonella typhimurium LT-2.

It has been determined from steady state kinetic studies using the sulfide ion selective electrode that O-acetylserine sulfhydrylase catalyzes a Bi Bi Ping Pong reaction between O-acetyl-L-serine and sulfide. Both O-acetyl-L-serine (OAS) and sulfide exhibit strong competitive substrate inhibition. A fit of all the data to the equation for the mechanism yields KOAS = 0.149 +/- 0.059 mM and KIOAS = 46.91 +/- 10.06 mM for O-acetyl-L-serine and KS2- = 0.066 +/- 0.004 mM and KIS2- = 0.013 +/- 0.006 mM for sulfide. Product inhibition studies varying either substrate at changing fixed levels of cysteine demonstrate that cysteine combines with enzyme at two places along the reaction sequence to produce inhibition with KiCys = 1.048 +/- 0.048 mM and KICys = 11.4 +/- 0.5 mM. Relatively high concentrations of acetate are required to produce inhibition and at least part of the acetate inhibition is due to ionic strength. However, the ability of acetate to reverse the spectral shift produced from the binding of O-acetyl-L-serine to enzyme and the isotope exchange between [14C]acetate and O-acetyl-L-serine does demonstrate that the O-acetyl-L-serine to acetate half-reaction is reversible. There is some doubt as to the specificity of acetate as a product inhibitor, since propionate can also be used to reverse the spectral shift. Spectral studies using ths spectral shift produced from binding O-acetyl-L-serine to enzyme confirms the assignment of a ping-pong mechanism since the spectral intermediate produced is alpha-aminoacrylic acid in Schiff base with pyridoxal phosphate and, therefore, the acetyl moiety has been beta eliminated. Isotope exchange has been demonstrated for both the O-acetyl-L-serine to acetate and sulfide to cysteine half-reactions which also confirms a ping-pong mechanism.

Cysteine↗