title: The substrate proton of the pyruvate kinase reaction.
abstract: The pyruvate kinase reaction occurs in separate phosphate- and proton-transfer stages: (formula; see text) K+, Mg2+, and Mg.ADP are known to be required for the phosphoryl transfer step, and K+ and Mg2+ with allosteric stimulation by MgATP are important for proton transfer. This paper uses the isotope trapping method with 3H-labeled water to identify the proton donor and determine when in the sequence of the catalytic cycle it is generated. When the enzyme was allowed to exchange briefly with 3H2O (pulse phase) and then diluted into a mixture containing PEP, ADP, and the cofactor K+, Mg2+, or Co2+ in D2O (chase phase), an amount of [3H]pyruvate was formed in great excess of the amount expected from steady-state catalysis in the diluted 3H-labeled water. With K+, Mg2+, and ADP at pH 6-9.5 in the pulse phase, a limit of 1.25 enzyme equiv of 3H were trapped. The concentration of PEP required for half-maximum trapping was 14-fold greater than its steady-state Km. Therefore, the rate constant for dissociation of the donor proton is estimated to be 14 times the steady-state rate of [3H]pyruvate formation, approximately 109 s-1, or 1500 s-1. At pD 6.4, Mg2+ and ADP were required in the chase, indicating that the ADP in the pulse was not bound tightly enough to be used in the chase. At pD 9.4, ADP was not required in the chase, only Mg2+ or Co2+, making it possible to limit the chase to one turnover from hybrid labeled complexes such as E.K.Mg.CoADP or E.K.Co.MgADP and PEP.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111.
authors.forename: I A
authors.initials: IA
authors.lastname: Rose
authors.forename: D J
authors.initials: DJ
authors.lastname: Kuo
mesh_terms: D000818:Animals; D001692:Biological Transport; D003903:Deuterium; D008274:Magnesium; D011188:Potassium; D011522:Protons; D011770:Pyruvate Kinase; D011817:Rabbits; D013379:Substrate Specificity
chemical_list: D011522:Protons; D003903:Deuterium; D011770:Pyruvate Kinase; D008274:Magnesium; D011188:Potassium