Kinetic and structural analysis of substrate specificity in two copper amine oxidases from Hansenula polymorpha

Cindy M. Chang, Valerie J. Klema, Bryan J. Johnson, Minae Mure, Judith P. Klinman, Carrie M. Wilmot

Research output: Contribution to journalArticle

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Abstract

The structural, underpinnings of enzyme substrate specificity are investigated in a pair of copper amine oxidases (CAOs) from Hansenula polymorpha (HPAO-1 and HPAO-2). The X-ray crystal structure (to 2.0 A resolution) and steady state kinetic data of the second copper amine oxidase (HPAO-2) are presented for comparison to those of HPAO-1. Despite 34% sequence identity and superimposable active site residues implicated in catalysis, the enzymes vary considerably in their substrate entry channel. The previously studied CAO, HPAO-1, has a narrow substrate channel. In contrast, HPAO-2 has a wide funnel-shaped, substrate channel, which also contains a side chamber. In addition, there are a number of amino acid, changes within the channels of HPAO-2 and HPAO-1 that may sterically impact the ability of substrates to form covalent Schiff base catalytic intermediates and to initiate chemistry. These differences can partially explain the greatly different substrate specificities as characterized by kcat/Km value differences. In HPAO-1, the kCat/Km for methylamine is 330-fold greater than for benzylamine, whereas in HPAO-2, it is benzylamine that is the better substrate by 750-fold. In HPAO-2, an inflated D/kcat/K m(methylamine) in relation to DKcat/K m(benzylamine) indicates that proton abstraction has been impeded more than, substrate release. In HPAO-1, DkCat/K m(S) changes little with the slow substrate and indicates a similar increase in the energy barriers that control both substrate binding and subsequent catalysis. In neither case is kcat/Km for the second substrate, O2, significantly altered. These results reinforce the modular nature of the active sites of CAOs and show that multiple factors contribute to substrate specificity and catalytic efficiency. In HPAO-1, the enzyme with, the smaller substrate binding pocket, both initial substrate binding and proton loss are affected by an increase in substrate size, while in HPAO-2, the enzyme with the larger substrate binding pocket, the rate of proton loss is differentially affected when a phenyl substituent in the substrate is reduced to the size of a methyl group.

Original languageEnglish (US)
Pages (from-to)2540-2550
Number of pages11
JournalBiochemistry
Volume49
Issue number11
DOIs
StatePublished - Mar 23 2010
Externally publishedYes

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Amine Oxidase (Copper-Containing)
Pichia
Substrate Specificity
Structural analysis
Protons
Kinetics
Substrates
Enzymes
Catalysis
Catalytic Domain
Schiff Bases
X-Rays
Amino Acids
benzylamine
methylamine

ASJC Scopus subject areas

  • Biochemistry

Cite this

Chang, C. M., Klema, V. J., Johnson, B. J., Mure, M., Klinman, J. P., & Wilmot, C. M. (2010). Kinetic and structural analysis of substrate specificity in two copper amine oxidases from Hansenula polymorpha. Biochemistry, 49(11), 2540-2550. https://doi.org/10.1021/bi901933d

Kinetic and structural analysis of substrate specificity in two copper amine oxidases from Hansenula polymorpha. / Chang, Cindy M.; Klema, Valerie J.; Johnson, Bryan J.; Mure, Minae; Klinman, Judith P.; Wilmot, Carrie M.

In: Biochemistry, Vol. 49, No. 11, 23.03.2010, p. 2540-2550.

Research output: Contribution to journalArticle

Chang, CM, Klema, VJ, Johnson, BJ, Mure, M, Klinman, JP & Wilmot, CM 2010, 'Kinetic and structural analysis of substrate specificity in two copper amine oxidases from Hansenula polymorpha', Biochemistry, vol. 49, no. 11, pp. 2540-2550. https://doi.org/10.1021/bi901933d
Chang, Cindy M. ; Klema, Valerie J. ; Johnson, Bryan J. ; Mure, Minae ; Klinman, Judith P. ; Wilmot, Carrie M. / Kinetic and structural analysis of substrate specificity in two copper amine oxidases from Hansenula polymorpha. In: Biochemistry. 2010 ; Vol. 49, No. 11. pp. 2540-2550.
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