Effects of Substrates on the Heat Stability and on the Reactivities of Thiol Groups of 3‐Phosphoglycerate Kinase

Pig muscle 3‐phosphoglycerate kinase contains seven cysteine residues/molecule enzyme. Two of them react with Ellman's reagent (Nbs 2 ) in a second‐order reaction [ k = (1.1 ± 0.1) × 10 3 M −1 s −1 ]; the reaction of the other five thiols are limited by a first‐order protein structural change [ k = (2.0 ± 0.4) × 10 −4 s −1 ] in 0.1 M Tris/HCl buffer, pH 7.5 at 20°C. Blocking the rapidly reacting thiols with Nbs 2 inactivated the enzyme (these two –SH groups are not equivalent in this respect), but it does not abolish substrate‐binding ability. The rapidly reacting thiol groups readily participate in intermolecular disulfide formation following their partial blocking with Nbs 2 . This type of aggregation of 3‐phosphoglycerate kinase molecules also leads to inactivation. The order of effectivity of substrates in inhibiting the reaction of the slowly reacting thiols is very similar to the order of their protective effect against heat inactivation. Both phenomena presumably reflect the structure‐stabilizing effect of substrates.