Abstract
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.
Highlights
Three point mutants of rabbit muscle PK (RMPK) were constructed using site-directed mutagenesis
Sixteen differences were found between the published nucleotide sequence and these clones (Fig. 2), twelve of which do not result in a change in the amino acid sequences, and four differences in the nucleotide sequence led to three differences in the amino acid sequence (Fig. 2)
The deduced amino acid sequence of the RMPK gene is different from the sequence reported by Larsen et al [32] in three residues
Summary
Materials—Pyruvate kinase from rabbit muscle, lactate dehydrogenase, disodium salt of ADP, phosphoenolpyruvate, Tris base, and TrisHCl were purchased from Boehringer Mannheim. The double-stranded DNA resulting from step 3 contained part of the coding and the noncoding sequence of 5Ј end of the RMPK gene. In step 2, double-stranded cDNA was synthesized by PCR amplification, employing the universal adapter primer, UAP (Life Technologies, Inc.), and the gene-specific primer P3 (5Ј-GTCCAGGAGGCCTGGGCT). Subcloning and DNA Sequencing—Based on the 5Ј and 3Ј end sequences obtained with the RACE methods, a set of primers (P5, 5ЈCTAAGAATTCATGTCGAAGTCCCACAGTGA-3Ј; P6, 5Ј-CTAAGAATTCATCGGTGGCACACTACAGC-3Ј) was generated bearing both the Nand C-terminal ends of the RMPK coding sequence flanked by EcoRI sites These primers were used to generate a 1.6-kilobase PCR fragment containing the entire open reading frame, which was subcloned into the multiple cloning site of pKK223-3 (Pharmacia Biotech Inc.). Where Vmax is the maximal velocity of each data set, [S] is the concentration of the variable substrate, n is the Hill coefficient, and Kapp is a complex steady-state kinetic equilibrium constant that is equivalent to the Km in the Michaelis-Menten equation where n ϭ 1
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
