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D B Keech

Publications and source records attributed to D B Keech.

At least 37 records · Page 2Linked to original sources
PubMed1976

Pyruvate carboxylase: affinity labelling of the magnesium adenosine triphosphate binding site.

The 2' , 3'-dialdehyde derivative of ATP (oATP) was prepared by periodate oxidation and on the following criteria was considered to be an effective affinity label. The magnesium complex of this derivative (Mg-oATP2) was shown to ba linear competitive inhibitor with respect to MgATP2-in both the acetyl-CoA-dependent and -independent activities of the enzyme but was a non-competitive inhibitor with respect to bicarbonate, and an uncompetitive inhibitor with respect to pyruvate. Mg-oATP was covalently bound to pyruvate carboxylase by reduction using sodium borohydride with concurrent irreversible inactivation of the enzyme...

Acetyl Coenzyme A↗
PubMed1975

Sheep kidney pyruvate carboxylase. Studies on the coupling of adenosine triphosphate hydrolysis and CO2 fixation.

Initial velocity and isotope exchange studies confirmed that the over-all reaction, like that catalyzed by pyruvate carboxylase purified from rat liver and chicken liver, was a nonclassical Ping Pong Bi Bi Uni Uni sequence with ATP and HCO3-binding randomly in the Bi Bi partial reaction. Three possible mechanisms for the coupling of ATP hydrolysis and CO2 fixation are considered: (i) Mechanism i, a concerted mechanism without the formation of a kinetically significant or detectable intermediate; (ii) Mechanism ii, activation of the enzyme by ATP to form an activated phosphoenzyme complex which can react with HCO3- by formation of a phosphorylated intermediate. On the basis of other evidence, an activated intermediate containing the ADP moiety was considered improbable. Evidence is presented which indicates that an isotopic exchange between ATP and ADP in the absence of added orthophosphate is not a property of the sheep kidney enzyme. This observation removed the need to postulate either that this exchange is an abortive reaction, or that there is a compulsory formation of a phosphoenzyme intermediate. Two analogues of ADP, alpha,beta-methylene adenosine diphosphate, and adenosine 5'-phosphosulfate, have been used to provide further evidence against Mechanism ii. Both compounds were competitive inhibitors with respect to MgATP2- (Ki values respectively, 0.58 mM and 3.0 mM, compared with 0.17 mM for ADP), but neither could be phosphorylated by the enzyme. Neither analogue could replace ADP in the HCO3-: oxalacetate isotopic exchange reaction, indicating that phosphorylation of ADP is necessary for this exchange to occur, and that Mechanism ii is not applicable. Since Mechanism iii involves formation of a carbonly phosphate intermediate, analogues of this compound, namely, carbamyl phosphate and phosphonacetic acid were used to examine this pathway. The fact that the enzyme catalyzed the synthesis of ATP from ADP and carbamyl phosphate, and that phosphonacetic acid was a noncompetitive inhibitor with respect to MgATP2- (Ki = 0.5 mM) provides strong evidence that a carbonyl phosphate derivative is involved in the reaction mechanism. However, we have not found from initial velocity studies evidence for the formation of this intermediate, and it may therefore have only a transient existence in an essentially concerted reaction.

Adenosine Diphosphate↗
PubMed1975

A kinetic approach to defining the role of chemically modifiable residues at the active sites of enzymes.

Initial velocity studies can be used to define the function of chemically modifiable residues in the active site of a multisubstrate enzyme. Since the method relies on measuring biological activity, it has the advantage that it can be used with small amounts of relatively impure enzyme, but requires that modified enzyme molecules have some residual catalytic activity. The kinetic analysis of the modified enzyme can be carried out in the presence of some unmodified enzyme molecules. Consideration of examples of single- and multisubstrate enzymic reactions shows that interpretation of changes in apparent Km and apparent V values following chemical modification, in terms of effects on substrate binding or catalysis or both, requires a detailed knowledge of the kinetic reaction sequence. In the case of multisubstrate enzymes, it is necessary to ensure that, during the kinetic investigation, the concentrations of the non-varied substrates remain at near-saturating levels. For this reason, and because modification may induce changes in the rate-limiting step, a full kinetic analysis of the modified enzyme is advisable.

Amino Acids↗