Slow binding of metal ions to pigeon liver malic enzyme: A general case

Pigeon liver malic enzyme was inhibited by lutetium ion through a slow-bind ing process, which resulted in a concave down tracing of the enzyme activit y assay. The fast initial rates were independent of lutetium ion concentrat ion, while the slow steady-state rates decreased with increasing Lu3+ conce ntration. The observed rate constant for the transition from initial rate t o steady-state rate, k(obs), exhibited saturation kinetics as a function of Lu3+ concentration, suggesting the involvement of an isomerization process between two enzyme forms (R-form and T-form). The binding affinity of Lu3 to the R-form is weaker (K-d,K-Lu = 14 muM) than that of Mn2+ (K-m,K-Mn = 1.89 muM); however, Lu3+ has much tighter binding affinity with the T-form (K-d,K-Lu* = 0.83 muM). Lu3+ was shown to be a competitive inhibitor with r espect to Mn2+, which suggests that Lu3+ and Mn2+ are competing for the sam e metal binding sire of the enzyme. These observations are in accordance wi th the available crystal structure information, which shows a distorted act ive site region of the Lu3+-containing enzyme. Other divalent cations, i.e, , Fe2+, Cu2+, Or Zn2+, also act as time-dependent slow inhibitors for malic enzyme. The dynamic quenching constants of the intrinsic fluorescence for the metal-free and Lu3+-containing enzymes are quite different, indicating the conformational differences between the two enzyme forms. The secondary structure of these two enzyme forms, on the other hand, was not changed. Th e above results indicated that replacement of the catalytically essential M n2+ by other metal ions leads to a slow conformational change of the enzyme and consequently alters the geometry of the active site. The transformed e nzyme conformation, however, is unfavorable for catalysis. Both the chemica l nature of the metal ion and its correct coordination in the active site a re essential for catalysis.