Send to:

Choose Destination
See comment in PubMed Commons below
Biochemistry. 2006 Apr 11;45(14):4683-94.

Relationship of stopped flow to steady state parameters in the dimeric copper amine oxidase from Hansenula polymorpha and the role of zinc in inhibiting activity at alternate copper-containing subunits.

Author information

  • 1Department of Chemistry and Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.


The expression of a copper amine oxidase (CAO) from Hansenula polymorpha in Saccharomyces cerevisiae under differing culture conditions leads to the incorporation of varied levels of CAO-bound zinc. The presence of substantial amount of zinc results in two distinctive enzyme species, designated as the fast and slow enzymes. Both forms are rapidly reduced by substrate methylamine with a rate constant of 199 s(-1) but behave remarkably differently in their oxidation rates; the fast enzyme is oxidized by dioxygen at a rate of 22.1 s(-1), whereas the slow enzyme reacts at a rate of 1.8 x 10(-4) s(-1). The apparent kcat of the enzyme preparation is linearly proportional to the fraction of the fast enzyme, with an extrapolated value of 6.17 s(-1) when the enzyme is 100% in its "fast" form. A comparison of rate constants for cofactor reduction and reoxidation steps, measured in stopped flow experiments, to the extrapolated kcat implicates additional steps in the steady state reaction. Measurement of the proportion of oxidized (ETPQ(ox)) and reduced cofactor (ETPQ(red)) under steady state conditions indicates approximately 50% of each cofactor form at 0.8 or 2 mM methylamine. Kinetic isotope effect measurements using deuterated amine substrate lead to the following steady state values: (D)(k(red)) = 8.5 (0.5), (D)(kcat) = 1.7 (0.1), and (D)(kcat/K(m)) = 4.3 (0.2). The collective data allow the calculation of partially rate-determining constants during the reductive half-reaction (ca. 200 s(-1) for binding of substrate to ETPQ(ox) and 27.9 s(-1) for release of aldehyde product or a protein isomerization from ETPQ(red)); an additional step with a rate constant of 13.2 s(-1) is assigned to the oxidative half-reaction, most likely for the release of product hydrogen peroxide. These results, together with the sole detection of oxidized and reduced cofactor during rapid scanning stopped flow experiments, indicate that four steps contribute to kcat, with the first electron transfer from cofactor to O2 contributing ca. 29%. An investigation of the relationship between the copper content and the extent of the fast enzyme shows that only the copper-containing homodimer is capable of rapid reoxidation and that zinc-copper heterodimers are incapable of rapid turnover at either subunit. This implies communication between the metal sites of the two subunits per dimer that impacts O2 binding and/or electron transfer from reduced cofactor to bound O2.

[PubMed - indexed for MEDLINE]
PubMed Commons home

PubMed Commons

How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for American Chemical Society
    Loading ...
    Write to the Help Desk