The Regulation Of The Nadp-specific Malic Enzyme In Escherichia Coli K12

Date of Award

1982

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Abstract

A purification procedure for the NADP-specific malic enzyme (E.C. 1.1.1.40) of Escherichia coli was developed and produced a homogenous NADP-ME of specific activity of 140 units per mg. The apparent molecular weights obtained by sedimentation-equilibrium centrifugation and disc gel electrophoresis were 470,000 and 478,000 respectively. The subunit molecular weight obtained by NaDodSO(,4)-gel electrophoresis was 80,500. It was concluded that the NADP-ME is a hexameric protein composed of identically sized subunits.;Both Mg('2+) and Mn('2+) fulfill the divalent cation requirement for NADP-ME activity but the high affinity of the enzyme for Mn('2+) and NADP('+) indicates that Mn('2+) is the preferred cofactor in vitro. The velocity-malate saturation curves are complex, indicative of combined apparent positive and negative cooperativity. Hill plot analysis of the malate-saturation data suggested that OAA and NADH caused different conformational changes in the enzyme. In the absence or presence of effectors, the malate-binding site interactions vary significantly with the choice of metal cofactor. The kinetic data can be explained by a model involving sequential ligand-induced conformational changes in the enzyme. Proteolytic digestion studies using homogenous NADP-ME support a model for distinct metal cofactor determined conformational states. These results substantiate the proposal that metal cofactors have a regulatory function.;Initial velocity-kinetic studies demonstrated that potassium activated the NADP-ME at concentrations less than 50 mM but was inhibitory at higher concentrations, in the presence of Mn('2+) or Mg('2+). Proteolytic and kinetic studies indicated a direct binding of K('+) to the NADP-ME resulting in changes in enzyme conformation and activity.;Solutions of high ionic strength strongly inhibited the NADP-ME activity and decreased the affinity for its substrates, probably through the stabilization of an unfavourable enzyme conformation. The acetate ion specifically activates the enzyme and desensitizes it to all effectors and regulation by Mn('2+) and Mg('2+).;Initial-velocity kinetic studies performed at varying ionic strengths and in the presence of acetate suggest that the malate, Mg('2+)- and NADP('+)-binding sites are independent and equivalent in the absence of all effectors. However, functional equivalence (physiological conditions) exists only between the metal cofactor binding sites.

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