Kinetic and Mutational Studies of the Number of Interacting Divalent Cations Required by Bacterial and Human Methionine Aminopeptidases

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• 作者：Xiaoyi V. Hu ; Xiaochun Chen ; Kee Chung Han ; Albert S. Mildvan ; Jun O. Liu
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：November 6, 2007
• 年：2007
• 卷：46
• 期：44
• 页码：12833 - 12843
• 全文大小：201K
• 年卷期：v.46,no.44(November 6, 2007)
• ISSN：1520-4995

文摘

Methionine aminopeptidases (MetAP) are responsible for the proteolytic removal of the initiatormethionine from nascent proteins. This processing permits multiple posttranslational modifications andprotein turnover. We have cloned, expressed in Escherichia coli, and purified the recombinant humanmitochondrial MetAP isoform (MetAP1D). The full-length enzyme and a truncated form lacking themitochondrial targeting sequence (residues 1-55) have been characterized as metal-requiring proteases,with Co²⁺ being the best activator. At the optimal pH (8.0), the k_cat of MetAP1D of 0.39 min^-1 is 280-fold lower, and the K_m of the substrate Met-Pro-p-nitroanilide (576 M) is 3-fold greater, than the respectivekinetic parameters obtained with MetAP from E. coli, although MetAP1D is 61% homologous to E. coliMetAP and their circular dichroic spectra are nearly identical. MetAP1D thus appears to be a less efficientenzyme than other known MetAPs in vitro. At saturating substrate concentrations, a plot of V_max versusfree Co²⁺ shows sigmoidal metal activation of MetAP1D, both with and without an N-terminal His-tag,with a Hill coefficient (n) of 1.9 and a K_0.5 of 0.40 M. Similarly, E. coli MetAP shows n = 2.1 and K_0.5= 0.2 M. Hence, at least two Co²⁺ ions, which may act cooperatively, are needed to promote catalysis,providing kinetic evidence for the functioning of both Co²⁺ ions of the binuclear complex found in theX-ray structure of E. coli MetAP [Roderick, S. L. and Matthews, B. W. (1993) Biochemistry 32, 3907-3912] and resolving a disagreement in the literature. The X-ray structure of the human cytosolic MetAP1showed three Co²⁺ ions at the active site, with the third Co²⁺ coordinated by the conserved residue His212 [Addlagatta, A., Hu, X., Liu, J. O., and Matthews, B. W. (2005) Biochemistry 44, 14741-14749].Consistent with the structure, kinetic studies of the human cytosolic MetAP1 yielded a Hill coefficient(n) of 2.9 and a K_0.5 of 0.26 M for activation by Co²⁺, as well as a k_cat of 25.5 min^-1 and a K_m of 740M for the substrate Met-Pro-p-nitroanilide. The H212A mutation decreased n to 2.2, decreased k_cat 60-fold to 0.42 min^-1, and increased K_0.5 6.5-fold to 1.8 M. The H212K mutation further decreased n to1.4, decreased k_cat 1800-fold to 0.014 min^-1, and increased K_0.5 158-fold to 41 M. Hence, at least threeCo²⁺ ions are needed to promote optimal catalysis by human MetAP1. Both mutations of His212 abolishedthe binding and/or the cooperativity of the third Co²⁺ ion, as indicated by the decreases in n and theincreases in K_0.5 of the remaining two Co²⁺ ions, but did not affect the K_m of the substrate. The moredamaging effects of the H212K mutation on both the Hill coefficient for Co²⁺ binding and the catalysissuggest that Lys 212 might directly compete with Co²⁺ for the third metal-binding site. Together, theseresults suggest that human MetAP1 is distinct from other members of the MetAP superfamily in thenumber of metal ions employed and likely mechanism of catalysis.