Biochemical Characterization of Human SET and MYND Domain-Containing Protein 2 Methyltransferase

摘要

SET and MYND domain-containing protein 2 (SMYD2) is a proteinnlysine methyltransferase that catalyzes the transfer of methyl groups from S-adenosylmethioninen(AdoMet) to acceptor lysine residues on histones and other proteins. Tonunderstand the kinetic mechanism and the function of individual domains, humannSMYD2 was overexpressed, purified, and characterized. Substrate specificity and productnanalysis studies established SMYD2 as a monomethyltransferase that prefers nonmethylatednp53 peptide substrate. Steady-state kinetic and product inhibition studies showednthat SMYD2 operates via a rapid equilibrium random Bi Bi mechanism at a rate ofn0.048 ( 0.001 su00011, with KMs for AdoMet and the p53 peptide of 0.031 ( 0.01 μM andn0.68(0.22 μM, respectively. Metal analyses revealed that SMYD2 contains three tightlynbound zinc ions that are important for maintaining the structural integrity and catalytic activity of SMYD2. Catalytic activity was alsonshown to be dependent on the GxG motif in the S-sequence of the split SET domain, as a G18A/G20A double mutant and ansequence deletion within the conserved motif impaired AdoMet binding and significantly decreased enzymatic activity. Thenfunctional importance of other SMYD2 domains including the MYND domain, the cysteine-rich post-SET domain, and thenC-terminal domain (CTD), were also investigated. Taken together, these results demonstrated the functional importance of distinctndomains in the SMYD family of proteins and further advanced our understanding of the catalytic mechanism of this family.