The Steady-State Kinetics of the Enzyme Reaction Tested by Site-Directed Mutagenesis of Hydrophobic Residues (Val, Leu, and Cys) in the C-Terminal α-Helix of Human Adenylate Kinase1.

To elucidate whether the C-terminal region in human adenylate kinase participates in the interaction with the substrate (MgATP2− and/or AMP2−), hydrophobic residues (Val-182, Vall86, Cysl87, Leul93, and Leul93) were substituted by site-directed mutagenesis and the steady-state kinetics of fifteen mutants were analyzed. A change in the hydro-phobic residues in the C-terminal domain affects the affinity for substrates (Km), that is, not only for MgATP2− but also for AMP2−, and the catalytic efficiency (kcat. The results obtained have led to the following conclusions: (i) Val182 may interact with both MgATP2− and AMP2− substrates, but to a greater extent with MgATP2−, and play a role in catalysis. (ii) Vall86 appears to play a functional role in catalysis by interacting with both MgATP2- and AMP2− to nearly the same extent, (iii) Cysl87 appears to play a functional role in catalysis, (iv) Leu 190 appears to interact with both MgATP2− and AMP2− substrates but to a greater extent with AMP2−, (v) Leul93 appears to interact with both MgATP2− and AMP2− but to a greater extent with AMP2−. 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 MgATP2− but also AMP2− 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 α-helixappears to interact with AMP, as if it has swung out and rotated to cover the adenine moieties. The C-terminal α-helix of human adenylate kinase appears to be essential for the interaction with adenine substrates by swinging out during catalysis. [ABSTRACT FROM AUTHOR]