title: Binding of dioxygen to non-metal sites in proteins: exploration of the importance of binding site size versus hydrophobicity in the copper amine oxidase from Hansenula polymorpha.
abstract: Copper amine oxidases (CAOs) contain 2,4,5-trihydroxyphenylalanyl quinone (TPQ) and a copper ion in their active sites, catalyzing amine oxidation to aldehyde and ammonia concomitant with the reduction of molecular oxygen to hydrogen peroxide. Kinetic studies on the CAO from bovine serum (BSAO) [Su and Klinman (1999) Biochemistry 37, 12513-12525] and the recent reports on the cobalt substituted form of the enzyme from Hansenula polymorpha (HPAO) [Mills and Klinman (2000) J. Am. Chem. Soc. 122, 9897-9904, and Mills et al. (2002) Biochemistry, 41, 10577-10584] support pre-binding of molecular oxygen prior to a rate-limiting electron transfer from the reduced form of TPQ (p-aminohydroquinone form) to dioxygen. Although there is significant sequence homology between BSAO and HPAO, k(cat)/K(m)(O2) for BSAO under the optimal condition is one order of magnitude lower than that for HPAO. From a comparison of amino acid sequences for BSAO and HPAO, together with the X-ray crystal structure of HPAO, a plausible dioxygen pre-binding site has been identified that involves Y407, L425, and M634 in HPAO; the latter two residues are altered in BSAO to A490 and T695. To determine which of these residues plays a greater role in dioxygen chemistry, k(cat)/K(m)(O2) was determined in HPAO for the M634 --> T and L425 --> A mutants. The L425 --> A mutation does not alter k(cat)/K(m)(O2) to a large extent, whereas the M634 --> T decreased k(cat)/K(m)(O2) by one order of a magnitude, creating a catalyst that is similar to BSAO. A series of mutants at M634 (to F, L, and Q) were, therefore, prepared in HPAO and characterized with regard to k(cat)/K(m)(O2) as a function of pH. Structure reactivity correlations show a linear relationship of rate with side chain volume, rather than hydrophobicity, indicating that dioxygen reactivity increases with the bulk of the residue at position 634. This site also shows specificity for O2, in relation to the co-gas N2, since substitution of the inert gas N2 by either Ar or He has no effect on measured rates. In particular, He gas is expected to have little affinity for protein at 1 atmospheric pressure, implying little or no binding by N2 as well.
authors.affiliation: Department of Chemistry, University of California, Berkeley, California 94720, USA.
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Goto
authors.forename: Judith P
authors.initials: JP
authors.lastname: Klinman
mesh_terms: D006631:Amine Oxidase (Copper-Containing) / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D003035:Cobalt / Q000378:metabolism*; D003300:Copper / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004295:Dihydroxyphenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*; D010843:Pichia / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D013329:Structure-Activity Relationship; D014783:Viscosity
chemical_list: C086758:2,4,5-trihydroxyphenylalanine quinone; D017931:DNA Primers; D003035:Cobalt; D004295:Dihydroxyphenylalanine; D003300:Copper; D006631:Amine Oxidase (Copper-Containing); D010100:Oxygen