Abstract
Abstract An alternative method of purifying 3-phosphoglycerate kinase (EC 2.7.2.3) from yeast is described which employs step-induced pH-gradient elution from phosphocellulose and electrofocusing. The purified enzyme has a specific activity of 1200 to 1400 units per mg when corrected to saturating conditions and 30°. Reaction of this enzyme with Woodward's reagent K. (N-ethyl-5-phenylisoxazolium-3'-sulfonate) leads to rapid inhibition; MgATP affords protection against inactivation. Inhibition obeys pseudo-first order kinetics and there is an enzyme inhibitor complex formed prior to covalent modification. Although up to 5 moles of carboxyl per mole of phosphoglycerate kinase can be modified by Woodward's reagent K, it appears that only a single carboxyl is essential for the enzyme's action. The essential carboxyl appears to be more reactive than the nonessential but modifiable carboxyls. Sedimentation velocity and optical rotatory dispersion studies indicate that there has been no major conformational change concomitant with modification. Possible mechanistic roles for the essential carboxyl are discussed.
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