Histone H3 lysine 9 (H3K9) methylation is a major component of gene regulation 
and chromatin organization. SUV39H1 methylates H3K9 at the pericentric 
heterochromatin region and participates in the maintenance of genome stability. 
In this study, a recombinant purified SUV39H1 is used for substrate specificity 
and steady-state kinetic analysis with peptides representing the un- or 
dimethylated lysine 9 histone H3 tail or full-length human recombinant H3 (rH3). 
Recombinant SUV39H1 methylated its substrate via a nonprocessive mechanism. 
Binding of either peptide or AdoMet first to the enzyme made a catalytically 
competent binary complex. Product inhibition studies with SUV39H1 showed that 
S-adenosyl-l-homocysteine is a competitive inhibitor of S-adenosyl-l-methionine 
and a mixed inhibitor of substrate peptide. Similarly, the methylated peptide 
was a competitive inhibitor of the unmethylated peptide and a mixed inhibitor of 
AdoMet, suggesting a random mechanism in a bi-bi reaction for recombinant 
SUV39H1 in which either substrate can bind to the enzyme first and either 
product can release first. The turnover numbers (k(cat)) for the H3 tail peptide 
and rH3 were comparable (12 and 8 h(-)(1), respectively) compared to the value 
of 1.5 h(-)(1) for an identical dimethylated lysine 9 H3 tail peptide. The 
Michaelis constant for the methylated peptide (K(m)(pep)) was 13-fold lower 
compared to that of the unmethylated peptide. The Michaelis constants for AdoMet 
(K(m)(AdoMet)) were 12 and 6 microM for the unmethylated peptide substrate and 
rH3, respectively. A reduction in the level of methylation was observed at high 
concentrations of rH3, implying substrate inhibition. Deletion of the 
chromodomain or point mutation of the conserved amino acids, W64A or W67A, of 
SUV39H1 impaired enzyme activity despite the presence of an intact catalytic SET 
domain. Thus, SUV39H1 utilizes both the chromodomain and the SET domain for 
catalysis.