Kinetic and mutational studies of the number of interacting divalent cations required by bacterial and human methionine aminopeptidases

Methionine aminopeptidases (MetAP) are responsible for the proteolytic removal of the initiator methionine from nascent proteins. This processing permits multiple posttranslational modifications and protein turnover. We have cloned, expressed in Escherichia coli, and purified the recombinant human mitochondrial MetAP isoform (MetAP1D). The full-length enzyme and a truncated form lacking the mitochondrial 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 MetAPID of 0.39 min^-1 is 280-fold lower, and the K_m of the substrate Met-Pro-p-nitroanilide (576 μM) is 3-fold greater, than the respective kinetic parameters obtained with MetAP from E. coli, although MetAPID is 61% homologous to E. coli MetAP and their circular dichroic spectra are nearly identical. MetAPID thus appears to be a less efficient enzyme than other known MetAPs in vitro. At saturating substrate concentrations, a plot of V _max versus free 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 μM. Similarly, E. coli MetAP shows n = 2.1 and K_0.5 = 0.2 μM. 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 the X-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 MetAP1 showed three Co²⁺ ions at the active site, with the third Co²⁺ coordinated by the conserved residue His 212 [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 MetAP 1 yielded a Hill coefficient (n) of 2.9 and a K_0.5 of 0.26 μM for activation by Co²⁺, as well as a k_cat of 25.5 min ^-1 and a K_m of 740 μM 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 μM. The H212K mutation further decreased n to 1.4, decreased k_cat 1800-fold to 0.014 min^-1, and increased K _0.5 158-fold to 41 μM. Hence, at least three Co²⁺ ions are needed to promote optimal catalysis by human MetAP1. Both mutations of His212 abolished the binding and/or the cooperativity of the third Co ²⁺ ion, as indicated by the decreases in n and the increases in K_0.5 of the remaining two Co²⁺ ions, but did not affect the K_m of the substrate. The more damaging effects of the H212K mutation on both the Hill coefficient for Co²⁺ binding and the catalysis suggest that Lys 212 might directly compete with Co²⁺ for the third metal-binding site. Together, these results suggest that human MetAP1 is distinct from other members of the MetAP superfamily in the number of metal ions employed and likely mechanism of catalysis.