Biomedical subjects

W P Jencks

Publications and source records attributed to W P Jencks.

At least 55 records · Page 3Linked to original sources

PubMed1986

Two functional domains of coenzyme A activate catalysis by coenzyme A transferase. Pantetheine and adenosine 3'-phosphate 5'-diphosphate.

Studies of the reactivity of succinyl-CoA:3-keto acid CoA transferase with a small coenzyme A analog, methylmercaptopropionate, have shown that noncovalent interactions between the enzyme and the side chain of CoA are responsible for a rate acceleration of approximately 10(12), which is close to the total rate acceleration brought about by the enzyme (Moore, S. A., and Jencks, W. P. (1982) J. Biol. Chem. 257, 10893-10907). We report here that interaction between the enzyme and the pantetheine moiety of CoA provides the majority of the rate acceleration and destabilization of the enzyme-thiol ester intermediate that is observed with CoA substrates. The role of the adenosine 3'-phosphate 5'-diphosphate moiety of CoA is to provide 6.9 kcal/mol of binding energy in order to pull the pantetheine moiety into the active site. The enzyme-thiol ester intermediate, E-pantetheine, was generated by reaction of pantetheine with the thiol ester of enzyme and methylmercaptopropionate. E-Pantetheine undergoes hydrolysis with khyd = 2 min-1, 140-fold faster than E-CoA, and reacts with acetoacetate with kAcAc = 3 X 10(6) M-1 min-1, only 10-fold slower than E-CoA. However, in the reverse direction acetoacetylpantetheine reacts with CoA transferase (kAcAc-SP = 220 M-1 min-1) 1.6 X 10(6) times slower than acetoacetyl-CoA. The equilibrium constant for the reaction of pantetheine with E-CoA is approximately 8 X 10(-6).

Acetoacetates↗

PubMed1984

Adenosine 5'-triphosphate at the active site accelerates binding of calcium to calcium adenosinetriphosphatase.

The complex of Mg X ATP and the calcium adenosinetriphosphatase of sarcoplasmic reticulum (E X ATP) reacts with 50-300 microM Ca2+ to form phosphoenzyme (E-P X Ca2) with a rate constant of 70 s-1 (pH 7.0, 100 mM KCl, 5 mM MgSO4, 25 degrees C, and SR vesicles passively loaded with Ca2+). This rate constant is independent of Ca2+ concentration above 50 microM. It is 4-6 times faster than the rate constants of 11-15 s-1 for the conformational change associated with Ca2+ binding in the absence of activation by ATP. The reaction of 200 microM Ca2+ with enzyme preincubated in 0.9 microM [gamma-32P]ATP X Mg shows a burst of [32P]E-P X Ca2 formation. This result indicates that Mg X ATP bound to the active site, and not a regulatory site, can accelerate the conformational change associated with Ca2+ binding because this concentration of Mg X ATP is well below the Kd of 160-500 microM for the putative regulatory site. When an unlabeled ATP chase is added with the Ca2+ to enzyme preincubated with [gamma-32P]ATP X Mg, the amount of [32P]E-P X Ca2 that is formed increases with the concentration of ATP in the preincubation solution and is consistent with a maximum fraction trapped of 0.55 and Kd = 4.5 microM for the dissociation of Mg X ATP from the active site. The fact that labeled E X ATP can be trapped by added Ca2+ confirms the conclusion that dissociation of ATP from E X ATP X Ca2 is slow relative to phosphorylation.

Adenosine Triphosphate↗

PubMed1984

Energetics of the calcium-transporting ATPase.

A thermodynamic cycle for catalysis of calcium transport by the sarcoplasmic reticulum ATPase is described, based on equilibrium constants for the microscopic steps of the reaction shown in Equation 1 under a single set of experimental (formula; see text) conditions (pH 7.0, 25 degrees C, 100 mM KCl, 5 mM MgSO4): KCa = 5.9 X 10(-12) M2, K alpha ATP = 15 microM, Kint = 0.47, K alpha ADP = 0.73 mM, K'int = 1.7, K"Ca = 2.2 X 10(-6) M2, and Kp = 37 mM. The value of K"Ca was calculated by difference, from the free energy of hydrolysis of ATP. The spontaneous formation of an acylphosphate from Pi and E is made possible by the expression of 12.5 kcal mol-1 of noncovalent binding energy in E-P. Only 1.9 kcal mol-1 of binding energy is expressed in E X Pi. There is a mutual destabilization of bound phosphate and calcium in E-P X Ca2, with delta GD = 7.6 kcal mol-1, that permits transfer of phosphate to ADP and transfer of calcium to a concentrated calcium pool inside the vesicle. It is suggested that the ordered kinetic mechanism for the dissociation of E-P X Ca2, with phosphate transfer to ADP before calcium dissociation outside and phosphate transfer to water after calcium dissociation inside, preserves the Gibbs energies of these ligands and makes a major contribution to the coupling in the transport process. A lag (approximately 5 ms) before the appearance of E-P after mixing E and Pi at pH 6 is diminished by ATP and by increased [Pi]. This suggests that ATP accelerates the binding of Pi. The weak inhibition by ATP of E-P formation at equilibrium also suggests that ATP and phosphate can bind simultaneously to the enzyme at pH 6. Rate constants are greater than or equal to 115 s-1 for all the steps in the reaction sequence to form E-32P X Ca2 from E-P, Ca2+ and [32P]ATP at pH 7. E-P X Ca2 decomposes with kappa = 17 s-1, which shows that it is a kinetically competent intermediate. The value of kappa decreases to 4 s-1 if the intermediate is formed in the presence of 2 mM Ca2+. This decrease and inhibition of turnover by greater than 0.1 mM Ca2+ may result from slow decomposition of E-P X Ca3.

Animals↗

PubMed1982

Reactions of acyl phosphates with carboxylate and thiol anions. Model reactions for CoA transferase involving anhydride formation.

Succinyl phosphate reacts rapidly with the thiol N-acetyl-beta-mercaptoethylamine (a CoA analog) to form the succinate thiol ester. This is a chemical model for the enzymic reaction of CoA transferase. It is demonstrated that: 1) the chemical reaction is stepwise, proceeding through the formation of a succinic anhydride intermediate. The rate-determining step at pH 6.6 is anhydride formation. 2) The apparent equilibrium constant for anhydride formation from succinyl phosphate is increased 6000-fold in going from pH 6.6 to 4.4. Formation of the reactive anhydride intermediate is so favorable at pH 4.4 that it accumulates in the reaction solution. 3) The rate constant for anhydride formation is accelerated 60-fold in going from pH 6.6 to 4.4 due to protonation of the phosphate moiety of succinyl phosphate, which enhances its leaving ability. 4) Anhydride formation through intramolecular reaction of the --COO- group of succinyl phosphate is accelerated greater than or equal to 10(5)-fold compared to the intermolecular reaction of 1 M acetate and acetyl phosphate. Thus, in the chemical reaction, anhydride formation can be made rapid and favorable by: 1) making the reacting groups intramolecular; and 2) catalyzing the expulsion of the leaving group. These are modes of rate acceleration that can occur in enzymic reactions. This model study provides support for a mechanism that proceeds through an anhydride intermediate in the enzymic reaction catalyzed by CoA transferase.

Acyltransferases↗

PubMed1982

Model reactions for CoA transferase involving thiol transfer. Anhydride formation from thiol esters and carboxylic acids.

Rate and equilibrium constants were determined for anhydride formation from the thiol esters p-nitrophenyl thiosuccinate (PTS) and N-acetyl-S-succinyl-beta-mercaptoethylamine, and from p-nitrophenyl thioacetate in the presence of substituted acetates. These thiol esters undergo thiol transfer reactions through rate-determining anhydride formation that are models for the enzymic reaction catalyzed by CoA transferase. The same value of beta nuc approximately equal to 1.0 for the nucleophilic reactions of substituted acetates with p-nitrophenyl thioacetate and with enzyme-CoA supports the anhydride mechanism for the enzymic reactions. The bimolecular reaction of succinyl-CoA with enzyme is 10(13) times faster than the nonenzymic reaction of acetyl-CoA with acetate; the intramolecular reaction of bound substrate is accelerated by a factor of kcat/kN - 10(11) M. The nonenzymic intramolecular reaction of PTS is faster than the nucleophilic reaction of acetate with acetyl-CoA by a similar factor of 4.5 X 10(10) M, of which 6 X 10(5) M is caused by intramolecularity and 7 X 10(4) by the increased chemical reactivity of PTS compared to acetyl-CoA. The enzyme may utilize binding energy to increase the reaction rate by making the reaction intramolecular (decreasing the requirement for loss of entropy) and by destabilizing the bound substrate relative to the transition state in an analogous manner. An identical rate increase of 6 X 10(5) M from intramolecularity was observed for the reaction of N-acetyl-S-succinyl-beta-mercaptoethylamine compared with the corresponding acetate. The rate increase in the intramolecular reaction of PTS reflects more favorable values of T delta S identical = 5.0 +/- 1.4 kcal mol-1 and -delta H identical to = 2.9 +/- 1.3 kcal mol-1 compared with the bimolecular reaction.

Acyltransferases↗

PubMed1982

Formation of active site thiol esters of CoA transferase and the dependence of catalysis on specific binding interactions.

The enzyme-CoA intermediate of succinyl-CoA:3-ketoacid coenzyme A transferase reacts with methyl mercaptopropionate, a short chain analog of coenzyme A, to release coenzyme A and give a new thiol ester at the active site, enzyme-SR (E-SR). The reaction is reversible, with Keq = 2.4 X 10(-4). Other thiols react similarly, acetyl-CoA. On the other hand, the value of kcat/km for the reaction of succinyl-SR with the enzyme to form E-SR is smaller than that for the reaction of succinyl-CoA to form enzyme-CoA by a factor of 3 X 10(12). Thus, interactions of the enzyme with the nonreacting portions of coenzyme A are responsible for a rate increase of 3 X 10(12) compared with the short chain analog. This increase is close to the total rate acceleration of 5 X 10(13) that is brought about by the enzyme. The rate increase is interpreted as representing 17 kcal mol-1 of intrinsic is interpreted as representing 17 kcal mol-1 of intrinsic binding energy of coenzyme A that is utilized to stabilize the transition state of the reaction; the observed binding of coenzyme A to the enzyme is relatively weak. The thiol ester group of enzyme-CoA is activated for reaction with the nonspecific reagents water, borohydride anion, and thiol anions by approximately 10(2)-10(3) compared with E-SR and thiol esters of acetate. The formation of E-SR at equilibrium is unfavorable by 5.7 kcal mol-2 compared with thiol esters of acetate, in spite of its normal chemical reactivity. This is consistent with a thermodynamically unfavorable conformation change upon thiol ester formation. The observed more favorable formation of enzyme-CoA compared to E-SR by 4.9 kcal mol-1 can be accounted for by utilization of the binding energy of coenzyme A to overcome the unfavorable Gibbs energy of this conformation change, for which there is independent evidence. The reaction of succinate with E-SR is 10(5) faster than with a thiol ester of acetate. This rate increase represents utilization of the binding energy of the specific substrate succinate for catalysis; however, no binding of succinate to E-SR was observed. A rate acceleration of 8 X 10(5) was observed for the nonphysiological reaction of coenzyme A with E-SR to give enzyme-CoA compared to the analogous nonenzymic reaction. This represents utilization of the binding energy of coenzyme A, in large part to overcome the entropy barrier for reaction. The combined effects of several modest contributions, especially the entropy loss of bound substrates, appear sufficient to account for most of the observed rate increase of the enzymic reaction without invoking ordinary mechanisms of chemical catalysis.

Acyl Coenzyme A↗

PubMed1982

Slow dissociation of ATP from the calcium ATPase.

The acyl-phosphate intermediate of the sarcoplasmic reticulum calcium ATPase reaction, formed in a brief incubation of vesicular enzyme with 5 microM [gamma-32P]ATP and calcium, reacts biphasically with added ADP (pH 7.0, 25 degrees C, 100 mM KCl, 5 mM MgSO4). Both the burst size and the rate constant for the slow phase increase with increasing ADP concentration in the way that is expected if the burst represents very rapid formation of an equilibrium amount of enzyme-bound ATP and the slow phase represents rate-limiting dissociation of ATP. Also consistent with this interpretation are the slow labeling of phosphoenzyme under conditions in which unlabeled ATP must dissociate first and the observation of a burst of ATP formation on ADP addition to phosphoenzyme. Values of the equilibrium constants for ADP dissociation from phosphoenzyme (0.75 mM), for ATP formation on the enzyme (2.3), and for the ATP dissociation rate constant (37 s-1) were obtained from a quantitative analysis of the data.

Adenosine Diphosphate↗

PubMed1981

Modification and inactivation of CoA transferase by 2-nitro-5-(thiocyanato)benzoate.

Succinyl-CoA:3-ketoacid coenzyme A transferase undergoes a biphasic reaction with 2-nitro-5-(thiocyanato)-benzoate, giving approximately 70% loss of activity in the initial phase. Active-site titration shows that this inactivation represents the complete loss of activity of 75 +/- 5% of the enzyme molecules. The remaining 25 +/- 5% of the active sites is protected against inactivation by methyl methanethiosulfonate and 5,5'-dithiobis(2-nitrobenzoate); this protection is removed upon treatment of the modified enzyme with dithiothreitol Values of kcat/Km for the two half-reactions catalyzed by the enzyme are the same for the native and modified enzymes on the basis of number of remaining active sites. The modified enzyme shows a smaller decrease in activity with increasing pH in the range pH 7.5-8.7 than the native enzyme. It is concluded that the "essential" thiol group of the enzyme is not involved directly in catalysis and that it reacts with 2-nitro-5-(thiocyanato)-benzoate by two pathways, to form active and inactive enzymes. This can be explained by the attack of the thiol on carbon to form active enzyme-SCN and the attack on sulfur to form enzyme-SSAr, which is blocked at the active site and rapidly undergoes irreversible inactivation.

Animals↗

PubMed1981

On the attribution and additivity of binding energies.

It can be useful to describe the Gibbs free energy changes for the binding to a protein of a molecule, A-B, and of its component parts, A and B, in terms of the "intrinsic binding energies" of A and B, DeltaG(A) (i) and DeltaG(B) (i), and a "connection Gibbs energy," DeltaG(s) that is derived largely from changes in translational and rotational entropy. This empirical approach avoids the difficult or insoluble problem of interpreting observed DeltaH and TDeltaS values for aqueous solutions. The DeltaG(i) and DeltaG(s) terms can be large for binding to enzymes and other proteins.

Journal Article↗

PubMed1980