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G L Kenyon

Publications and source records attributed to G L Kenyon.

At least 91 records · Page 5Linked to original sources
PubMed1977

Mandelate racemase from Pseudomonas putida. Absence of detectable intermolecular proton transfer accompanying racemization.

An equimolar mixture of DL-[alpha-2H]- and DL-[alpha-13C]mandelate, when incubated with mandelate racemase (EC 5.1.2.2), shows conversion of singly labeled mandelate to unlabeled mandelate, due to solvent exchange of the alpha proton, while the level of doubly labeled mandelate remains at a constant low level. Similarly, an equimolar mixture of unlabeled and DL-[alpha-2H,alpha-13C]mandelate, when incubated with the enzyme, shows conversion of doubly labeled mandelate to singly labeled mandelate, due to solvent exchange, while the level of unlabeled mandelate remains constant at 50%. Incubation of an equimolar mixture of DL-[alpha-3H]mandelate and DL-rho-chloromandelate. These results indicate that mandelate racemase does not catalyze an intermolecular proton transfer to achieve racemization. These data are necessary, but not sufficient, results to indicate that mandelate racemase operates via a one-acceptor mechanism, in which the proton abstracted from one stereochemical face of a substrate molecule is returned to the opposite face of the same carbon of the substrate molecule.

Carbon Isotopes↗
PubMed1977

Properties of a CH3-blocked creatine kinase with altered catalytic activity. Kinetic consequences of the presence of the blocking group.

Steady state kinetic parameters for rabbit muscle creatine kinase (EC 2.7.3.2) and this enzyme stoichiometrically blocked at the iodoacetamide-sensitive cysteinyl residue with a CH3S-group have been measured at 30+/-0.1 degrees, pH 9.00, using Mg(II) as the required metal ion cofactor. The double reciprocal plots for the CH3S-blocked enzyme with MgATP as the variable substrate are biphasic, each curve showing a break at approximately 1.9 mM MgATP, and suggest the possibility of negative cooperativity in metal-nucleotide binding. Furthermore, extrapolated lines at high MgATP concentrations intersect on the abscissa, indicating loss of synergism in binding of substrates. In contrast, observed Michaelis constants for creatine are, within experimental error, the same for both native and blocked enzymes. The maximal velocity of the CH3S-blocked enzyme is found to be 28.1% of the value of the native enzyme. Product inhibition patterns for both native and blocked enzyme are also compared. Again, these patterns indicate that the CH3S-blocking group alters the nucleotide binding site more than the guanidino substrate binding site. Calculations using the methods of Chou and Fasman (1970) Biochemistry 13, 211-222) lead to the prediction that the active cysteinyl residue occurs at the beginning of a beta turn which separates two portions of beta sheet structure of the enzyme, and so may be in a position to mediate conformational changes in the protein.

Adenosine Diphosphate↗
PubMed1975

Mandelate racemase from Pseudomonas putida. Magnetic resonance and kinetic studies of the mechanism of catalysis.

The interactions of mandelate racemase with divalent metal ion, substrate, and competitive inhibitors were investigated. The enzyme was found by electron paramagnetic resonance (EPR) to bind 0.9 Mn2+ ion per subunit with a dissociation constant of 8 muM, in agreement with its kinetically determined activator constant. Also, six additional Mn2+ ions were found to bind to the enzyme, much more weakly, with a dissociation constant of 1.5 mM. Binding to the enzyme at the tight site enhances the effect of Mn2+ on the longitudinal relaxation rate (1/T1p) of water protons by a factor of 11.9 at 24.3 MHz. From the frequency dependence of 1/T1p, it was determined that there are similar to 3 water ligands on enzyme-bound Mn2+ which exchange at a rate larger than or equal to 10-7 sec-1. The correlation time for enzyme-bound Mn2+-water interaction is frequency-dependent, indicating it to be dominated by the electron spin relaxation time of Mn2+. Formation of the ternary enzyme-Mn2+-mandelate complex decreases the number of fast exchanging water ligands by similar to 1, but does not affect tau-c, suggesting the displacement or occlusion of a water ligand. The competitive inhibitors D,L-alpha-phenylglycerate and salicylate produce little or no change in the enzyme-Mn2+-H2O interaction, but ternary complexes are detected indirectly by changes in the dissociation constant of the enzyme-Mn2+ complex and by mutual competition experiments. In all cases the dissociation constants of substrates and competitive inhibitors from ternary complexes determined by magnetic resonance titrations agree with K-M and K-i values determined kinetically and therefore reflect kinetically active complexes. From the paramagnetic effects of Mn2+ on 1/T1 and 1/T2 of the 13C-enriched carbons of 1-[13C]-D,L-mandelate and 2-[13C]-D,L-mandelate, Mn2+ to carboxylate carbon and Mn2+ to carbinol carbon distances of 2.93 plus or minus 0.04 and 2.71 plus or minus 0.04 A, respectively, were calculated, indicating bidentate chelation in the binary Mn2+-mandelate complex. In the active ternary complex of enzyme, Mn2+, and D,L-mandelate, these distances increase to 5.5 plus or minus 0.2 and 7.2 plus or minus 0.2 A, respectively, indicating the presence of at least 98.9% of a second sphere complex in which Mn2+, and C1 and C2 carbon atoms are in a linear array. The water relaxation data suggest that a water ligand is immobilized between the enzyme-bound Mn2+ and the carboxylate of the bound substrate. This intervening water ligand may polarize or protonate the carboxyl group. From 1/T2p the rate of dissociation of the substrate from this ternary complex (larger than or equal to 5.6 times 10-4 sec-1) is at least 52 times greater than the maximal turnover number of the enzyme (1070 sec-1), indicating that the complex detected by nuclear magnetic resonance (NMR) is kinetically competent to participate in catalysis. Relationships among the microscopic rate constants are considered.

Binding, Competitive↗
PubMed1975

Simple alkanethiol groups for temporary blocking of sulfhydryl groups of enzymes.

New reagents for the temporary blocking of active or accessible sulfhydryl groups of enzymes have been developed. These reagents, which are either alkyl alkanethiolsulfonates or alkoxycarbonylalkyl disulfides, rapidly and quantitatively place various RS- groups on the sulfhydryls to generate mixed disulfides. In all cases native enzymes can be regenerated with either dithiothreitol or beta-mercaptoethanol. In general the temporary blocking groups also afford total protection against normally inhibitory thiol blocking agents. When RS- groups were attached to rabbit muscle creatine kinase (EC 2.7.3.2), a trend toward lower residual activities with increasing bulk was observed. Treatment of the native creatine kinase with 14CH3HgC1 led to incorporation of greater than 1 equiv of CH3Hg- group per subunit. This CH3Hg- blocked enzyme was fully active, and the blocking group afforded no protection against iodoacetamide. These results suggest that CH3Hg- and the RS- groups are modifying two different sulhydryl groups on the enzyme. When papain (EC 3.4.4.10) was treated with excess methyl methanethiolsulfonate. complete and rapid inhibition was observed, and 1 equiv of CH3S- was incorporated/mol of active enzyme. Complete protection against normally inhibitory 5,5'-dithiobis(2-nitrobenzoic acid) was afforded by the temporary blocking group. When rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) was titrated with methyl methanethiolsulfonate, two sulfhydryl groups per subunit were found to be modified, one much more rapidly than the other. If one extrapolates the initial slope of the titration curve, the inactivation of the enzyme would be complete after modification of a single cysteinyl residue per subunit.

Alkanes↗