The kinetic properties of four isoforms of acid phosphatase purified from mature soybean (Glycine max) seeds were studied using glycolytic metabolites as substrates. The isoforms AP1, AP3A and AP3B presented maximal activities around pH 4.0, and AP2 at pH 6.0, for phosphoenolpyruvate (PEP) as a substrate. With glucose-6-P (G6P) and fructose-6-P (F6P) maximal activities were observed at pH 5.5 for the AP3A and AP3B isoforms. The acid phosphatases presented the following apparent K-m values, at the corresponding optimum pH values: AP1 (p-nitrophenylphosphate (pNPP): 0.49, PEP: 0.23 mM); AP2 (pNPP: 0.38, PEP: 0.47 mM); AP3A (pNPP: 0.20, PEP: 0.10, G6P: 0.30, F6P: 0.16 mM) and AP3B (pNPP: 0.086, PEP: 0.078, G6P: 0.31, F6P: 0.33 mM). Maximal specificity constant (V-max/K-m) was obtained for the isoform:AP1, with PEP as a substrate. Independent of the substrates, the reactions catalyzed by the soybean acid phosphatase isoforms were potently inhibited by molybdate and to a lesser extent by Zn2+. Inhibitions were also observed in the presence of fluoride, with PEP as a substrate, and by Cu2+, and p-chloromercuribenzoate (pCMB) with G6P and F6P as substrates. Our results suggest that the Four acid phosphatase forms could play important roles in plant metabolism, acting on key glycolytic intermediates. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.14714954