The steady-state kinetics of the enzyme reaction tested by site-directed mutagenesis of hydrophobic residues (Val, Leu, and Cys) in the C-terminal alpha-helix of human adenylate kinase.

Abstract

To elucidate whether the C-terminal region in human adenylate kinase participates in the interaction with the substrate (MgATP(2-) and/or AMP(2-)), hydrophobic residues (Val182, Val186, Cys187, Leu190, and Leu193) were substituted by site-directed mutagenesis and the steady-state kinetics of fifteen mutants were analyzed. A change in the hydrophobic residues in the C-terminal domain affects the affinity for substrates (K(m)), that is, not only for MgATP(2-) but also for AMP(2-), and the catalytic efficiency (k(cat)). The results obtained have led to the following conclusions: (i) Val182 may interact with both MgATP(2-) and AMP(2-) substrates, but to a greater extent with MgATP(2-), and play a role in catalysis. (ii) Val186 appears to play a functional role in catalysis by interacting with both MgATP(2-) and AMP(2-) to nearly the same extent. (iii) Cys187 appears to play a functional role in catalysis. (iv) Leu190 appears to interact with both MgATP(2-) and AMP(2-) substrates but to a greater extent with AMP(2-). (v) Leu193 appears to interact with both MgATP(2-) and AMP(2-) but to a greater extent with AMP(2-). The activity of all mutants decreased due to the change in substrate-affinity. The closer the residue is located to the C-terminal end, the more its mutation affects not only MgATP(2-) but also AMP(2-) substrate binding. The hydrophobic alterations disrupt hydrophobic interactions with substrates and that might destabilize the conformation of the active site. The more C-terminal part of the alpha-helix appears to interact with AMP, as if it has swung out and rotated to cover the adenine moieties. The C-terminal alpha-helix of human adenylate kinase appears to be essential for the interaction with adenine substrates by swinging out during catalysis.