Modelling of substrate binding to 3‐phosphoglycerate kinase with analogues of 3‐phosphoglycerate

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Abstract
Two short analogues of 3‐phosphoglycerate, −OOC‐CHOH‐CH2‐O‐PO2−3, phosphonolactate, (‐OOCCHOH‐CH2‐PO2−3) and arsonolactate (‐OOC‐CHOH‐CH2‐AsO32−3) 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‐PO2−3 and ‐OOC‐CHOHCH2‐CH2‐AsO2−3, 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°C, the inhibitory constants (K1) 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 fastreacting thiols. This comparison suggests that the shorter analogues bind differently, and that the catalytic mechanism demands a precise fitting of the ‐CH2‐O‐PO2−3 segment of the substrate.