Replacement of enzyme-bound calcium with strontium alters the kinetic properties of methanol dehydrogenase

T. K. Harris, V. L. Davidson

Research output: Contribution to journalArticlepeer-review

23 Scopus citations


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 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 activator and competitive inhibitor of methanol-dependent activity in native MEDH from P. denitrificans. Sr-MEDH exhibited a similar K(I) for cyanide inhibition of methanol-dependent activity, but the K(A) 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 and crystallographic studies that suggest an apparently unique role for Ca2+ in MEDH compared with other Ca2+-dependent proteins and enzymes.

Original languageEnglish (US)
Pages (from-to)175-182
Number of pages8
JournalBiochemical Journal
Issue number1
StatePublished - 1994
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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