l-Phenylalanine stereospecifically inhibits the renaturation of muscle pyruvate kinase |
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Authors: | David H Porter Janet M Cardenas |
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Institution: | Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27514 U.S.A. |
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Abstract: | Bovine type M pyruvate kinase can be reversibly denatured by solutions of guanidine HCl. Subsequent dilution of the enzyme into buffer containing β-mercaptoethanol or dithiothreitol results in recovery of enzymatic activity with an average half-time of 17 min at 16 °C. The addition of 1 mm l-phenylalanine increases the average half-time for recovery of enzymatic activity to 26 min, while 8 mm l-phenylalanine further increases this value to 46 min. Tyrosine and tryptophan also inhibit the reactivation but to a lesser extent than phenylalanine. Neither l-alanine, l-valine, d-phenylalanine, phosphoenolpyruvate, nor fructose 1,6-bisphosphate have any appreciable effect on activity recovery rates, either in the presence or absence of l-phenylalanine. Phenylpyruvate is a very potent inhibitor of reactivation. The addition of 5 mm phenylpyruvate increases the half-time to 57 min. The evidence presented in this paper supports the hypothesis that an l-phenylalanine-binding site which probably is distinct from the catalytic site is formed early in the renaturation process. l-Phenylalanine binds to this site and inhibits two first-order relaxations that are rate limiting for the reactivation and that have the following rate constants: 8.76 × 10?2 and 1.24 × 10?2 min?1, respectively, in the absence of phenylalanine and 3.04 × 10?2 and 7.63 × 10?3min?1, respectively, in the presence of 8.0 mm phenylalanine. We presume these first-order processes to be transconformational steps in the reactivation process. |
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