Modelling of substrate binding to 3-phosphoglycerate kinase with analogues of 3-phosphoglycerate.
Two short analogues of 3-phosphoglycerate, -OOC-CHOH-CH2-O-PO32-, phosphonolactate, (-OOC-CHOH-CH2-PO32-) and arsonolactate (-OOC-CHOH-CH2-AsO32-) have been tested with 3-phosphoglycerate kinase. None of these served as substrate for the kinase reaction, unlike the previously studied [Orr, G. A. & Knowles, J. R. (1974) Biochem. J. 141, 721-723] analogues -OOC-CHOH-CH2-CH2-PO32- and -OOC-CHOH-CH2-CH2-AsO32-, which are isosteric with 3-phosphoglycerate. Thus, a decrease in the substrate size and the accompanying stereochemical changes cannot be tolerated by the catalytic mechanism. Instead, both analogues acted as relatively poor competitive inhibitors with respect to both 3-phosphoglycerate and MgATP. AT pH 8.5 and 20 degrees C, the inhibitory constants (Ki) of phosphonolactate and arsnolactate against both substrates are 17 +/- 5 mM and 30 +/- 7 mM, respectively. Surprisingly, however, both analogues proved to be more effective than either 3-phosphoglycerate or its isosteric analogues in protecting the enzyme against modification of its fast-reacting thiols. This comparison suggests that the shorter analogues bind differently, and that the catalytic mechanism demands a precise fitting of the -CH2-O-PO32- segment of the substrate.
Vas
M
M
Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest.
D000818:Animals; D001152:Arsenicals / Q000494:pharmacology; D001665:Binding Sites; D005988:Glyceric Acids / Q000378:metabolism*; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D010735:Phosphoglycerate Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013552:Swine
D001152:Arsenicals; D005988:Glyceric Acids; D013438:Sulfhydryl Compounds; C005156:3-phosphoglycerate; D010735:Phosphoglycerate Kinase; C033355:arsonoacetic acid