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Rose IA , Kuo DJ
The substrate proton of the pyruvate kinase reaction
Biochemistry. 1989 ;28(25) :9579-9585
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Abstract
The pyruvate kinase reaction occurs in separate phosphate- and proton-transfer stages. 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 K(m). Therefore, the rate constant for dissociation of the donor proton is estimated to be 14 times the steady-state rate of [3H]pyruvate formation, ~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. The 3H trapped from these complexes are 1 and 1.8, respectively. With Co2+ and ADP in both the pulse and chase, 3 enzyme equiv of 3H were trapped. The value of 1.8 equiv trapped in one turnover is consistent with the preequilibration of a donor containing three 3H with the substrate, PEP, containing two 1H to produce pyruvate with three equivalent positions of hydrogen, i.e., 3/5 of 3 = 1.8. Muirhead et al. (1987) have concluded that Lys-269 is likely to be the proton donor on the basis of crystallographic studies of pyruvate kinase of cat muscle. In terms of lysine the amount of label trapped in pyruvate would depend on positional isotope exchange (PIX) to mix the three NH3+ protons in the ternary complexes and the extent of exchange that occurs between Lys-(?)NH2 and medium before the next recycle of reaction, steps 1 and 4, respectively. With Mg2+ step 1 would have to be slow and step 4 rapid compared with the rate of product formation to explain the apparent monoprotonic nature of the donor. The opposite would be true with Co2+, allowing 1.8 equiv to be trapped in one turnover and three in multiple turnovers. For the pulse and chase pHs to be as high as 9.5 without a decrease in the amount of 3H that could be trapped a lysine of very high pK(a) is required. To achieve proton-exchange rates >103 s-1 from a group of such high pK(a) requires catalysis. Buffer catalysis could be ruled out. Therefore, internal exchange probably occurs with a residue of low pK(a) with good access to the medium. The residue may be remote, requiring a proton relay, or it may be a residue in the active-site cavity such as Glu-271, which in the absence of PEP might have direct or indirect access to Lys-269. Upon addition of PEP this circuit would be broken as the Glu-271 serves some important function in the catalyses or is otherwise diverted.
Notes
00062960 (ISSN) Cited By: 9; Export Date: 30 May 2006; Source: Scopus CODEN: BICHA Language of Original Document: English Correspondence Address: Kuo, D.J.; Institute for Cancer Research; Fox Chase Cancer Center Philadelphia, PA 19111, United States Chemicals/CAS: proton, 12408-02-5, 12586-59-3; pyruvate kinase, 9001-59-6, Deuterium, 7782-39-0; Magnesium, 7439-95-4; Potassium, 7440-09-7; Protons; Pyruvate Kinase, EC 2.7.1.40