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 replaced with Sr2+ by growing the host bacterium, Paracoccus denitrificans, in media in which Ca2+ was replaced with Sr2+. MEDH, which was purified from these cells (Sr-MEDH), exhibited an increased absorption coefficient for the PQQ chromophore, and displayed certain kinetic properties which were different from those of native MEDH. Native MEDH exhibits an endogenous activity which is not stimulated by substrate and which is inhibited by cyanide. Sr-MEDH exhibited lower endogenous activity which was stimulated by substrate, and was much less sensitive to inhibition by cyanide. The Vmax. for the methanol-dependent activity of Sr-MEDH was 3-fold greater than that of the native enzyme, and the Ks for methanol was altered. Cyanide also acts as an obligatory activator 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 K1 for cyanide inhibition of methanol-dependent activity, but the KA for cyanide activation of this 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 that of the native enzyme. These data confirm and significantly extend the conclusions from genetic [Richardson and Anthony (1992) Biochem. J. 287, 709-715] and crystallographic [White, Boyd, Mathews, Xia, Dai, Zhang and Davidson (1993) Biochemistry 32, 12955-12958] studies that suggest an apparently unique role for Ca2+ in MEDH compared with other Ca(2+)-dependent proteins and enzymes.
Harris
T K
TK
Department of Biochemistry, University of Mississippi Medical Center, Jackson 39216-4505.
Davidson
V L
VL
D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D000641:Ammonia / Q000737:chemistry; D002118:Calcium / Q000378:metabolism*; D007700:Kinetics; D010231:Paracoccus denitrificans / Q000201:enzymology; D011190:Potassium Cyanide / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D013324:Strontium / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature
D000641:Ammonia; D000429:Alcohol Oxidoreductases; C019641:alcohol dehydrogenase (acceptor); D011190:Potassium Cyanide; D002118:Calcium; D013324:Strontium