Biomedical subjects

W J Ray

Publications and source records attributed to W J Ray.

At least 109 records · Page 6Linked to original sources

PubMed1976

Thermodynamics and mechanism of the PO3 transfer process in the phosphoglucomutase reaction.

The equilibria among the central complexes in the phosphoglucomutase system were evaluated by (a) using an excess of enzyme plus Mg2+ to prepare mixtures with glucose phosphates in which essentially no free glucose phosphates were present; (b) inactivating the enzyme in such mixtures by means of a procedure that prevents substantial interconversion of the central complexes; and (c) assaying the quenched mixture for glucose 1-P, glucose 1-6-P2, and glucose-6-P. The fractional amounts of Ep-Mg-Glc-1-P, ED-Mg-Glc-P2, and Ep-Mg-Glc-6-P present at pH 7.5 and 24 degrees C were 0.13, 0.54, and 0.33. (Ep and ED are the phospho and dephospho forms of the enzyme, respectively). From these fractions and the equilibrium isotope exchange constants for the three sugar phosphates, true dissociation constants can be calculated for each of the above complexes: 8.5 muM, 19 nM, and 57 muM, respectively. Relative to the rate of PO3 transfer to water, a 3 x 10(10)-fold rate increase is produced by binding glucose-1-P to the Mg2+-enzyme (Ray, jr., W.J., Long, J.W., and Owens, J.D. (1976), Biochemistry, the following paper in this issue). This "substrate-induced rate effect" is equivalent to a difference of some 14 kcal in Gibbs activation energies for transfer to chemically similar hydroxyl groups, and most of this energy difference ultimately must be rationalized in terms of binding interactions involving the phosphoglucosyl moiety. Three different mechanisms for using substrate binding energy to reduce the activation energy of the subsequent catalytic step are examined as possible explanations for the substrate-induced rate effect. These mechanisms emphasize (a) enthalpic destabilization and (b) (entropic) immobilization of reactant groups during formation of the enzyme-substrate complex, and (c) increased binding interactions of nonreactant groups during the subsequent approach to the transition state. As a test for enthalpic destabilization of the enzymic phosphate group, values of deltaG degrees' for the hydrolytic cleavage of this group in Ep and Ep-Glc-1-P are calculated from equilibria measured at pH 7.5 and 30 degrees C: about -1 and +1.4 kcal/mol, respectively. To test for destabilization of the acceptor hydroxyl group in the enzyme-substrate complex, deltaG degrees' for equilibrium, Ep-Glc-P in equilibrium ED-Glc-P2, is compared with that for the corresponding process involving the nonrigid acceptor, 1,4-butanediol monophosphate: about -0.9 and -1.9 kcal, respectively. These results are not consistent with a large enthalpic destabilization of the reactant groups in the Ep-Glc-1-P complex. To test for entropic immobilization of reactant groups, glucose-6-phosphate is considered as a bidentate ligand, and the chelate effect on the binding and subsequent enzymic transfer reaction that arises from covalently linked the sugar ring and the PO3 group is evaluated. Reference reactions involving xylose as a PO3 acceptor both in the presence and absence of bond (inorganic) phosphite are used...

Binding Sites↗

PubMed1976

An analysis of the substrate-induced rate effect in the phosphoglucomutase system.

The rate constant for the catalytic transfer of the active-site PO3 group from rabbit muscle phosphoglucomutase to the hydroxyl group of a water molecule is about 3 x 10(-8) s-1 under optimal reaction conditions, but in the absence of the normal substrate, viz., at pH 7.5 and 30 degrees C, in the presence of saturating Mg2+; the corresponding constant for transfer to the 6-hydroxyl group of glucose 1-phosphate under analogous conditions, about 1000 s-1, is larger than this by some 3 x 10(10)-fold. Since no single factor appears to be capable of providing a rationale for a majority of this "substrate-induced rate effect" (Ray, jr., W.J., and Long, J.W. (1976), Biochemistry, the preceding paper in this issue), the change in the PO3-transfer rate produced by binding various parts of the phosphoglucosyl moiety to the enzyme, both separately and concurrently, was investigated. The rate of PO3 transfer to water is increased by up to 1000-fold by binding entities that provide the active site with a second PO3 group, e.g., ethyl phosphate or inorganic phosphite. Using an alcoholic acceptor further increases transfer efficiency (in the presence of bound phosphite): increase with methanol, about 2000-fold on a molar basis. The reactivities of ten other primary aliphatic alcohols vary by nearly 600-fold as the acidity of the PO3 acceptor is varied over a 4000-fold range. Although no straightforward relationship is observed between the efficiency of an alcohol as an acceptor and its acidity - presumably because of complications due to steric effects, for example - an increased transfer rate of 100-fold, relative to the water reaction, is estimated for a simple primary alcohol with a pKa similar to that expected for the 6-hydroxyl group of glucose 1-phosphate, when the alcohol is present at a concentration of 1 M. Joining an alcoholic acceptor and a PO3 group via five apparently inert bridging units changes PO3 transfer to an intramolecular process; in the case of 1,4-butanediol monophosphate the rate of transfer also increases by 240-fold, relative to the analogous reaction in the presence of 1 M propanol and bound inorganic phosphite. Comparable values also are obtained in comparisons of PO3 transfer rates for trans- 1,4-butenediol and 1,4-butynediol monophosphates relative to 1 M allyl and propargyl alcohols, respectively, in the presence of bound phosphite. An increased rate of transfer also is produced by binding the xylosyl part of the glucose ring, either when the acceptor is an hydroxyl group attached to the ring or when it is the hydroxyl group of a water molecule, e.g., as in the water reaction facilitated by bound xylose 1-phosphate. These and other results suggest that most of the differences between the rates of the water reaction and the glucose 1-phosphate reaction can be rationalized in terms of four fairly discrete factors whose approximate values are as follows: the PO4 factor, 1000-fold; the C-OH/H-OH factor, 100-fold; the nucleophile-binding factor, 250-fold; and the (CHOH)3-bridging factor, 200-fold...

Binding Sites↗

PubMed1976

The thermodynamic and structural differences among the catalytically active complexes of phosphoglucomutase: metal ion effects.

When the identity of the metal ion activator, M, is changed within the series, Zn2+, Co2+, Mg2+, Ni2+, Mn2+, and Cd2+, the equilibrium distribution among the central complexes in the phosphoglucomutase system is markedly altered. (The central complexes are Ep-M-Glc-6-P, ED-M-Glc-1,6-P2, and Ep-M-Glc-1-P, where Ep and ED are the phospho and dephospho forms of the enzyme). This altered distribution is caused by a metal-specific change in the equilibrium constant for transfer of the enzymic PO3 group to bound glucose monophosphates: 65-fold as M is varied from Zn2+ to Cd2+. This change in equilibrium is related to metal-specific differences in chemical potential of the phosphate group in the Ep-M complex; these differences in chemical potential remain in the Ep-M-Glc-1-P and Ep-M-Glc-6-P complexes, but essentially disappear in the ED-M-Glc-1,6-P2 complex. If glucose monophosphates are considered as substrates, and glucose bisphosphate as the product, there is a direct relationship between the equilibrium concentration of enzyme-substrate and enzyme-product complexes (when these are varied by changing the identity of the bound metal ion) and the ultraviolet spectrum of the equilibrium mixture of complexes, as assessed by difference spectroscopy (Peck, E.J., Jr., and Ray, W.J., Jr. (1969), J. Biol, Chem. 244, 3754). These spectral changes apparently are caused by an alteration in the conformation of the enzyme during transfer of a PO3 group between the enzyme and the glucose phosphate moiety, or as the result of it. The extent to which conformational changes accompany group-transfer processes in other enzymic systems is not clear, but it is possible that analogous changes may help to account for the "half-of-the-sites reactivity" observed with a number of multimeric enzymes.

Binding Sites↗

PubMed1976