Metallo-��-lactamase (imp-1) Expression, Purification, Identification, And Inhibitor Dansylcnsh Edta Interaction Mechanism And Functional Analysis

Objective To establish the expression, purification and characterization methods of IMP-1 in E.coli BL21 and acquire high purified enzyme; to investigate the interaction mechanism of IMP-1 with inhibitor EDTA and measure the kinetic parameters; to synthesize the thiol inhibitors , and explore the inhibitive mechanism with IMP-1 and its mutant W28A and fluorescence features as well as the detection of IMP-1 using fluorescence probe DansylC2SH; to construct the IMP-1 mutants , measure the kinetic parameters of IMP-1 mutants with substrates interaction and explore their function.Methods The plasmid pET9a/d-IMP-l was transformed into Ecoli BL21,after over-expressed, the products were sonicated and the sonicated solutions were purified by SP-Sepharose Column and SP-G75 Column, the purified product was identified by SDS-PAGE , its molecular weight and purifity were determined by MALDI-TOF MASS and UV. Using Cephaloridine as substrate, study the inactivation of IMP-1 with inhibitor EDTA at different pH and temperature, the enzyme catalytic reaction rates were measured by the hydrolysis curve of Cephaloridine , and the activation energy Ea and entropy AS were calculated by Arrhenius equation . The new thiol inhibitors DansylCnSH were synthesized by chemical method , the fluorescence properties for the interaction of the inhibitors DansylCnSH with IMP-1 were explored and the dissociate constants were measured ; the relationship between FI with IMP-1 concentrations was established, using Nitrocefin as substrate, measure the hydrolysis curve of the interaction for IMP-1 and its mutant with substrate in the presence of inhibitor DansylCnSH calculate the inhibitive constant Ki by the enzyme catalytic reaction rate, detect the IMP-1 concentration inculture and extracted solution through the fluorescence probe DansylC2SH. Devise IMP-1 mutant primer, do the LA-PCR experiments using the plasmid pKF18/d-IMP-l as template, the purified PCR products were validated by determining their DNA sequences ,then transformed the plasmid into Ecoli JM109 , after over-expression, the sonicated solutions were purified by SP-Sepharose Column and SP-G75 Column. Measure the the kinetic parameters and CD spectra for the interaction of IMP-1 mutants with substrate and investigate the pH effects on the enzyme catalytic reaction rate.Results the IMP-1 was mainly concentrated on the number 29 to 40 tubes, the SDS-PAGE results showed that there were little the other proteins in the number 33,34,35 tubes, the IMP-1 molecular weight is 25111.9 by MALDI-TOF-MS, using Nitrocefin as substrate, the Michaelis constant for IMP-1 is 9.28umol/L. The inactivation of IMP-1 processes two steps in the presence of EDTA, namely fast reaction and slow reaction, the fast reaction and slow reaction rate constants kf, ks were 1.50, 0.085 th"1] respectively ,the activity ratio for mono-zinc and di-zinc enzyme was 0.69, the activation energy Ea and entropy AS for the inactivation reaction were 74.55KJ-mol"1, -0.46 J-mol"1 . the fluorescence intensity ratios for DansylCnSH to DansylCnSH with IMP-1 mixture were 3.0(DansylC2SH),3.0(DansylC3SH)5.8(DansylC4SH),3.4(DansylC5SH), 3.4 (DansylC6SH); the dissociation constants for IMP-1 with DansylCnSH were 356±14 ( DansylC2SH ) ^ 975±110 ( DansylC3SH ) . 67±14(DansylC4SH\ 154±41 (DansylC5SH\ 105±42 (DansylC6SH); the inhibition reaction mechanism for DansylCnSH was mix- competitive inhibition, assuming the mechanism is competitive inhibition, its inhibition constants were 0.79> 1.27 ^ 0.63 > 0.32 ^ 0.33; the inhibition reaction mechanism for DansylC4SH, DansylC5SH, DansylC6SH was competitiveinhibition ,the inhibition constants for DansylCnSH with IMP-1 mutant were3.13(DansylC4SH); 4.02(DansylC5SH); 2.83(DansylC6SH), and the inhibition reaction mechanism for DansylC2SH, DansylC3SH was mix-competitive inhibition. The amino acid sequences for IMP-1 mutants indicated that amino acid mutates from V—>I, V—>A, V—*G at 25 position, the mole ratio of Zinc to protein for IMP-1 mutants was 2.2(V25I),2.3(V25G)#! 2.0 (V25A) respectively, and the enzyme catalytic reaction kinetics parameters were listed as following table: A)WTAntibiotickcat / min"1Km /fj.Mkcat/Km /HM-W1Cephaloridine53009.0588.9Imipenem700039179.5Nitrocefin270009.32903.2penicillin G3400060056.7(B)V25IAntibiotickcat / min"1Km / (4.M^/Km /nM-W1Cephaloridine0.21700.0012Imipenem22034000.065Nitrocefin4602816.43penicillin G (C) V25G1312000.0108Antibiotickcat / min"1Km / |nMWKm /nM-Wn"1Cephaloridine152900.0517Imipenem1206900.17Nitrocefin171.412.14penicillin G 34 2900 0.0117...