Isoforms of Human O-GlcNAcase Show Distinct Catalytic Efficiencies

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Jing Li^*, Cai-luan Huang, Lian-wen Zhang, Lin Lin, Zhong-hua Li, Fu-wu Zhang, and Peng Wang^*

College of Pharmacy and State Key Laboratory of Element-Organic Chemistry, Nankai University, Tianjin 300071, China; fax: +86-22-2350-6290; E-mail: jinglink@nankai.edu.cn; pwang@nankai.edu.cn

^* To whom correspondence should be addressed.

Received March 29, 2010; Revision received May 21, 2010

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O-GlcNAcase (OGA) is a family 84 glycoside hydrolase catalyzing the hydrolytic cleavage of O-linked β-N-acetylglucosamine (O-GlcNAc) from serine and threonine residues of proteins. Thus far, three forms of OGA have been identified in humans. Here we optimized the expression of these isoforms in E. coli and characterized their kinetic properties. Using Geno 3D, we predicted that N-terminal amino acids 63-342 form the catalytic site for O-GlcNAc removal and characterized it. Large differences are observed in the K_m value and catalytic efficiency (k_cat/K_m) for the three OGA variants, though all of them displayed O-GlcNAc hydrolase activity. The full-length OGA had the lowest K_m value of 0.26 mM and the highest catalytic efficiency of 3.51·10³. These results reveal that the N-terminal region (a.a. 1-350) of OGA contains the catalytic site for glycoside hydrolase and the C-terminal region of the coding sequence has the ability to stabilize the native three-dimensional structure and further affect substrate affinity.

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KEY WORDS: O-GlcNAcase, isoform, substrate affinity, catalytic efficiency

DOI: 10.1134/S0006297910070175

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