REPLACEMENT OF ENZYME-BOUND CALCIUM WITH STRONTIUM ALTERS THE KINETIC-PROPERTIES OF METHANOL DEHYDROGENASE

Methanol dehydrogenase (MEDH) possesses tightly bound Ca2+ in addition to its pyrroloquinoline quinone (PQQ) prosthetic group. Ca2+ was repl aced with Sr2+ by growing the host bacterium, Paracoccus denitrificans , in media in which Ca2+ was replaced with Sr2+. MEDH, which was purif ied from these cells (Sr-MEDH), exhibited an increased absorption coef ficient for the PQQ chromophore, and displayed certain kinetic propert ies which were different from those of native MEDH. Native MEDH exhibi ts an endogenous activity which is not stimulated by substrate and whi ch is inhibited by cyanide. Sr-MEDH exhibited lower endogenous activit y which was stimulated by substrate, and was much less sensitive to in hibition by cyanide. The V-max. for the methanol-dependent activity of Sr-MEDH was 3-fold greater than that of the native enzyme, and the K- s for methanol was altered. Cyanide also acts as an obligatory activat or and competitive inhibitor of methanol-dependent activity in native MEDH from P. denitrificans [Harris and Davidson (1993) Biochemistry 32 , 4362-4368]. Sr-MEDH exhibited a similar K-I for cyanide inhibition o f methanol-dependent activity, but the K-A for cyanide activation of t his activity was 17-fold greater than that for the native enzyme. The activation energy of Sr-MEDH was 13.4 kJ (3.2 kcal)/mol lower than tha t of the native enzyme. These data confirm and significantly extend th e conclusions from genetic [Richardson and Anthony (1992) Biochem. J. 287, 709-715] and crystallographic [White, Boyd, Mathews, Xia, Dai, Zh ang and Davidson (1993) Biochemistry 32, 12955-12958] studies that sug gest an apparently unique role for Ca2+ in MEDH compared with other Ca 2+-dependent proteins and enzymes.