Identification of domain-domain docking sites within Clostridium symbiosumpyruvate phosphate dikinase by amino acid replacement

Potential domain-domain docking residues, identified from the x-ray structu re of the Clostridium symbiosum apoPPDK, were replaced by site-directed mut agenesis, The steady-state and transient kinetic properties of the mutant e nzymes were determined as a way of evaluating docking efficiency. PPDK muta nts, in which one of two stringently conserved docking residues located on the N-terminal domain (Arg(219) and Glu(271)) was substituted, displayed la rgely unimpeded catalysis of the phosphoenolpyruvate partial reaction at th e C-terminal domain, but significantly impaired catalysis (>10(4)) of the A TP pyrophosphorylation of His(455) at the N-terminal domain: In contrast, a lanine mutants of two potential docking residues located on the N-terminal domain (Ser(262) and Lys(149)), which are not conserved among the PPDKs, ex hibited essentially normal catalytic turnover. Arg(219) and Glu(271) were t hus proposed to play an important role in guiding the central domain and, h ence, the catalytic His(455) into position for catalysis, Substitution of c entral domain residues Glu(434)/Glu(437) and Thr(453), th, respective docki ng partners of Arg(219) and Glu(271) resulted in mutants impaired in cataly sis at the ATP active site. The x-ray crystal structure of the apo-T453A PP DK mutant was determined to test for possible misalignment of residues at t he N-terminal domain-central domain interface that might result from loss o f the Thr(453)-Glu(271) binding interaction. With the exception of the muta tion site, the structure of T453A PPDK was found to be identical to that of the wild-type enzyme. It is hypothesized that the two Glu(271) interfacial binding sites that remain in the T453A PPDK mutant, Thr(453) backbone NH a nd Met(452) backbone NH, are sufficient to stabilize the native conformatio n as observed in the crystalline state but may be less effective in populat ing the reactive conformation in solution.