Investigation of Substrate Specificity in Copper Amine Oxidases from Hansenula Polymorpha

A second copper amine oxidase from Hansenula polymorpha (HPAO-2) has been expressed, kinetically characterized and x-ray crystal structure solved. Steady state and transient state kinetics of HPAO-2 have been measured and compared to the first studied copper amine oxidase from H. polymorpha (HPAO-1). Complementary mutations have been made in the substrate binding channels of both enzymes and the mutants characterized. Herein is presented a comparative study of the determinants of substrate specificity in a pair of copper amine oxidases from H. polymorpha. Comparison of steady state kinetic measurements of HPAO-1 and HPAO-2 reveal a marked difference in substrate specificity. Differences in the kinetic isotope effect on the second order rate constant for substrate for preferred versus nonpreferred substrate indicate significant substrate discrimination at the proton abstraction step for HPAO-2. Conversely with HPAO-1, similar isotope effects on kcat/Km for both preferred and nonpreferred substrates coupled with large differences in kcat/Km for substrate suggest both substrate binding and proton abstraction are involved in determining specificity. Comparison of the x-ray crystal structures of HPAO-1 and HPAO-2 suggest that the narrower binding channel of HPAO-1 may sterically predispose the enzyme to react with small aliphatic amines. Transient state kinetics were performed to determine the extent to which the proton abstraction step is affected by change in substrate. Significant kinetic isotope effects with HPAO-1 vs. both preferred and nonpreferred substrates and HPAO-2 vs. nonpreferred substrate were observed along with huge differences in rates of proton abstraction between substrates. The data demonstrate that substrate specificity on both enzymes extend beyond substrate binding and derive in part from differences in rates of hydrogen abstraction. Complementary substrate binding channel mutants in HPAO-1 and HPAO-2 were kinetically characterized. A tyrosine residue in HPAO-1 in the substrate binding channel corresponds to a cysteine residue in HPAO-2. HPAO-1 Y323C mutant shows a significant change in substrate specificity, with changes in both kcat/Km and kC-H, relative to wild type. HPAO-2 C306Y mutant is less active overall with little change in substrate specificity. Additionally, an oxygen-dependent off reaction pathway intermediate is observed in C306Y upon reaction with methylamine.