Nucleotide binding to type-M1 pyruvate kinase from rabbit muscle

Guy, Edward Charles (1986) Nucleotide binding to type-M1 pyruvate kinase from rabbit muscle. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

The binding of a number of ligands to type-M₁ pyruvate kinase was investigated by the ligand protection method using TNBS as the alkylating inhibitor. ADP and ATP were found to bind to free enzyme with a K_a of 2.1mM and 1.7mM respectively. Investigations of the effect of pH on the enzyme-nucleotide interaction reveal the binding of ADP and ATP to be independent of proton concentration between pH 6.5 and 8. At higher values of pH the affinity of the enzyme for nucleotide decreases such that a plot of pK_a against pH has a slope of -1. This finding implies a single group to be involved in nucleotide binding, with a pK_a of approximately 8, located on either the substrate or the enzyme. Since none of the proton dissociation constants for ADP and ATP correspond with the values measured it appears likely that the observed pK_a of 8 is due to a group located on the enzyme. Mg²⁺ has a more complex effect on the TNBS-medicated rate of inactivation of pyruvate kinase than the simple protection exhibited by ADP and ATP. At pH 7.5 the rate of inactivation of the enzyme by TNBS increases with Mg²⁺-concentration up to approximately 1mM. At higher concentrations of Mg²⁺, however, protection is observed. NAD binding at pH 7.5 results in an increase in the rate of inactivation of enzyme by TNBS. The reactive triazine dye Procion Blue MX-R was found to be an irreversible inhibitor of type-M₁ pyruvate kinase. The inactivation profile of the enzyme by Procion Blue MX-R shows a biphasic curve in which approximately 45% of total enzyme activity is lost with the incorporation of 1 molecule of dye/monomer of enzyme. The remaining 55% of initial activity is lost in an apparently linear fashion with the incorporation of a further 3 molecules of dye into each monomer. Measurement of this profile in the presence of 30mM ADP indicates that the binding of free ADP competes only with the second phase of the interaction such that one additional molecule of dye/monomer is required for the complete inactivation of the enzyme. The inactivation profile measured in the presence of 30mM ADP and 15mM Mg²⁺ reveals that both the first and second phases of the interaction are blocked. The results of ligand protection studies using Procion Blue MX-R as the alkylating probe are similar to those obtained using TNBS. The ligand binding properties of pyruvate kinase partially inactivated with approximately 0.7 molecules of Procion Blue MX-R/monomer were investigated. Ligand protection studies show the dissociation constant of ADP from the modified enzyme at pH 7.4 to be 9.5mM. This implies that partial modification with dye reduces the affinity of the enzyme for ADP. However, neither K_m(ADP), nor the potentiating effect of NAD on the rate of enzyme inactivation is affected by partial modification of the enzyme with dye. An overall model is proposed in which distinct nucleotide binding sites exist on each monomer of type-M₁ pyruvate kinase. It is envisaged that ligand binding at one site can affect binding at other sites on the same, and adjacent enzyme monomers.