Effects of conserved residues on the regulation of rabbit muscle pyruvate kinase.

A cDNA encoding the complete rabbit muscle pyruvate kinase isozyme (RMPK) was cloned using the method of rapid amplification of cDNA ends. The sequence encodes a polypeptide chain of 530 amino acids which differs in three amino acid residues from a sequence reported by Larsen et al. (Larsen, T.M., Laughlin, T., Holden, H.M., Rayment, L, and Reed, G.H. (1994) Biochemistry 33, 6301-6309). Glu233-Gln234 and Ala400 were identified instead of Asp233-Glu234 and Ser400, respectively. The recombinant RMPK was overexpressed in the Escherichia coli JM 105 cells. Purified recombinant pyruvate kinase displayed identical physical and enzymatic properties as the authentic enzyme. Three point mutants of RMPK were constructed using site-directed mutagenesis. Like the wild type RMPK, sedimentation, and CD spectroscopic studies show that purified RI 19C and T340M are tetrameric proteins with similar secondary and tertiary structures. Mutant R119C enzyme exhibits 0.6% of the value of k(cat) and an order of magnitude decrease in the apparent affinity for ADP as compared to the wild type PK. The overall response to inhibitor and activator, Phe and FBP, respectively, were not affected by the R119C mutation. The T340M mutant enzyme is only half as active as the wild type PK. T340M is more susceptible to inhibition by Phe but apparently is not responsive to the activator FBP. The kinetic behavior of the Q377K mutant enzyme is in between that of the R119C and T340M mutants exhibiting 5% of the wild type enzymatic activity and an enhanced sensitivity to the inhibitor, Phe, while maintaining the same responsiveness to FBP and apparent affinities for substrates. The significant decrease in activity in all three mutants mimics the exact consequences of the same mutations in human erythrocyte PK from hemolytic anemia patients. Thus, this study demonstrates not only the effects of these conserved residues in the regulatory properties of mammalian PK. but also that the observed effects are most likely applicable to all isozymic forms of PK.