Sequential structural changes of Escherichia coli thioesterase/protease I in the serial formation of Michaelis and tetrahedral complexes with diethyl p-nitrophenyl phosphate.
Escherichia coli thioesterase/protease I (TEP-I) belongs to a new subclass of lipolytic enzymes of the serine hydrolase superfamily. Here we report the first direct NMR observation of the formation of the Michaelis complex (MC) between TEP-I and diethyl p-nitrophenyl phosphate (DENP), an active site directed inhibitor of serine protease, and its subsequent conversion to the tetrahedral complex (TC). NMR, ESI-MS, and kinetic data showed that DENP binds to TEP-I in a two-step process, a fast formation of MC followed by a slow conversion to TC. NMR chemical shift perturbation further revealed that perturbations were confined mainly to four conserved segments comprising the active site. Comparable magnitudes of chemical shift perturbations were detected in both steps. The largest chemical shift perturbation occurred around the catalytic Ser(10). In MC, the conformation of the mobile Ser(10) was stabilized, and its amide resonance became observable. From the large chemical shift perturbation upon conversion from MC to TC, we propose that the amide protons of Ser(10) and Gly(44) serve as the oxyanion hole proton donors that stabilize the tetrahedral adduct. The pattern of residues perturbed in both steps suggests a sequential, stepwise structural change upon binding of DENP. The present study also demonstrates the important catalytic roles of conserved residues in the SGNH family of proteins.
Tyukhtenko
Sergiy I
SI
Institute of Biomedical Sciences, Academia Sinica, Nankang, Taipei, Taiwan 11529, Republic of China.
Litvinchuk
Alexandra V
AV
Chang
Chi-Fon
CF
Lo
Yu-Chih
YC
Lee
Shin-Jye
SJ
Shaw
Jei-Fu
JF
Liaw
Yen-Chywan
YC
Huang
Tai-Huang
TH
D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008245:Lysophospholipase / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D010261:Paraoxon / Q000737:chemistry*; D033903:Periplasmic Proteins / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D021241:Spectrometry, Mass, Electrospray Ionization
D029968:Escherichia coli Proteins; D033903:Periplasmic Proteins; D008245:Lysophospholipase; C400724:tesA protein, E coli; D010261:Paraoxon