Structure-function relationships in 3-phosphoglycerate kinase: role of the carboxy-terminal peptide.
Yeast 3-phosphoglycerate kinase (PGK) is a monomeric enzyme (Mr approximately 45,000) composed of two globular domains. Each domain corresponds approximately to the amino- and carboxy-terminal halves of the polypeptide chain. The carboxy-terminal end extends over the interdomain "hinge" region and packs against the amino-terminal domain. It has been proposed that domain movement, resulting in closure of the active site cleft, is essential for the catalytic function of PGK. Large-scale conformational changes have also been postulated to explain activation of the enzyme by sulfate ions. Using site-specific mutagenesis, we have removed a 15-amino-acid carboxy-terminal fragment, in order to probe its role in the substrate- and sulfate-induced conformational changes. The truncated enzyme exhibited approximately 1% of the activity of native PGK and lost the ability to undergo sulfate-induced activation. The Km for ATP was essentially unchanged (Km = 0.23 mM) in comparison to the native enzyme (Km = 0.30 mM), whereas the Km value for 3-phosphoglycerate was increased about eightfold (Km = 3.85 mM and 0.50 mM, respectively). These results suggest that the carboxy-terminal segment is important for the mechanism of the substrate- and sulfate-induced conformational transitions. CD spectra and sedimentation velocity measurements indicate that the carboxy-terminal peptide is essential for structural integrity of PGK. The increased susceptibility of the truncated enzyme to thermal inactivation implies that the carboxy-terminal peptide also contributes to the stability of PGK.
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Division of Biology, Beckman Research Institute of the City of Hope, Duarte, California 91010.
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D002384:Catalysis; D002942:Circular Dichroism; D003198:Computer Simulation; D007700:Kinetics; D009154:Mutation; D010455:Peptides / Q000502:physiology*; D010735:Phosphoglycerate Kinase / Q000302:isolation & purification / Q000502:physiology*; D015202:Protein Engineering; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013329:Structure-Activity Relationship; D013431:Sulfates / Q000494:pharmacology; D013816:Thermodynamics; D014461:Ultracentrifugation
D010455:Peptides; D013431:Sulfates; D010735:Phosphoglycerate Kinase