| Summary: | 博士 === 東海大學 === 生物學系 === 92 === Leucine aminopeptidases (LAPs) are exopeptidases that remove the N-terminal L-leucine from peptide substrates. For expression of Bacillus stearothermophilus NCIB 8924 leucine aminopeptidase II (LAP II) in Escherichia coli regulated by a T5 promoter, the gene was amplified by polymerase chain reaction and cloned into expression vector pQE-32 to generate pQE-LAPII. The His6-tagged enzyme was overexpressed in IPTG-induced E. coli M15 (pQE-LAPII) as a soluble protein and was purified to homogeneity by nickel-chelate chromatography to a specific activity of 425 U/mg protein with a final yield of 76%. The subunit molecular mass of the purified protein was estimated to be 44.5 kDa by SDS-PAGE. The temperature and pH optima for the purified protein were 60oC and 8.0, respectively. Under optimal condition, the purified enzyme showed a marked preference for Leu-ρ-nitroanilide, followed by Arg- and Lys-derivatives. The His6-tagged enzyme was stimulated by Co+2 ions, but was strongly inhibited by Cu+2 and Hg+2 and by the chelating agents, DTT and EDTA. The EDTA-treated enzyme could be reactivated with Co+2 ions, indicating it is a cobalt-dependent exopeptidase. Taking the biochemical characteristics together, we found that the recombinant LAPII (rLAPII) exhibits significant similarities from those properties described for the native enzyme.
Oxidative stability assay showed that the rLAPII was sensitive to oxidative damage by hydrogen peroxide at the elevated temperature. The H2O2-treated enzyme experienced obvious changes in the secondary structure when the oxidant concentration increased to 300 mM. To investigate the role of methionine residues on the oxidative inactivation, each of the five methionine residues in the rLAPII was replaced with leucine by site-directed mutagenesis. The mutant enzymes with an apparent Mr of approximately 44.5 kDa. The specific activities for Met82Leu, Met88Leu, Met254Leu, and Met382Leu were similar to that of the wild-type enzyme, whereas a reduced activity was observed in Met136Leu. The 50% decrease in the catalytic efficiency (kcat/Km) for Met136Leu was caused by 47% decrease in kcat value. As compared with the wild-type enzyme, all mutant proteins were more sensitive to the oxidant, implying that the methionine residues of B. stearothermophilus LAP II are important for the protection of the enzyme from oxidative inactivation.
Metallopeptidases form the most diverse of the catalytic types of peptidases, about 33 families being recognized. LAP is a member of family M29 of metallopeptidases, and the metal-binding residues remain unknown. In order to identify the Co2+-binding domain, we performed the alignment of amino acid sequence of LAPs from different microorganisms to determine the conserved amino acid residues, which might be essential for enzyme activity. The conserved amino acid residues of rLAPII include Asp-61, Asp-180, Asp-308, Asp-339, Asp-380, Asp-391, Asp-396, His-191, His-227, His-345 and His-378 were changed by site-directed mutagenesis. Replacement of 7 Asp residues with Ala and 4 His residues with Leu. There is no enzymatic activity were detected in Asp380Ala, His345Leu and His378Leu. Co2+ affinity of Asp61Ala was decreased obviously. The Km values for the other mutants were increased more than 30% by comparison with wild-type enzyme. The kcat values for Asp391Ala, Asp396Ala, His191Leu and His227Leu were increased, while kcat decreased 40 to 60%for Asp339Ala, Asp61Ala and Asp308Ala. The 83% decreased in the catalytic efficiency (kcat/Km) for Asp61Ala and the 70% decreased was exhibited by Asp308Ala and Asp339Ala. Alterations in Asp-180, Asp-391, Asp-396, His-191 and His-227 did not cause a significant difference on the catalytic efficiency. The EDTA-treated enzyme experienced obvious reactivation in the wild-type, Asp180Ala, Asp308Ala, Asp339Ala, Asp391Ala, Asp396Ala, His191Leu and His227Leu by CoCl2 addition. The significant reactivation of EDTA-treated Asp61Glu was observed, while the reactivation of EDTA-treated Asp61Ala was feebly. Moreover, apo-Asp380Ala, apo-His345Leu, apo-His345Arg, apo-His378Leu and apo-His378Arg could not restore the LAP activity by CoCl2 addition. Based on these results, it is suggested that the carboxyl group of Asp-61 may be play an important role in cobalt-binding, while Asp-380, His-345 and His-378 were the putative essential residues for enzyme structure.
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