Linked Life Data

17929833

Source:http://linkedlifedata.com/resource/pubmed/id/17929833

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
44
pubmed:dateCreated
2007-10-30
pubmed:abstractText
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 kcat of MetAP1D of 0.39 min-1 is 280-fold lower, and the Km of the substrate Met-Pro-p-nitroanilide (576 microM) is 3-fold greater, than the respective kinetic parameters obtained with MetAP from E. coli, although MetAP1D is 61% homologous to E. coli MetAP and their circular dichroic spectra are nearly identical. MetAP1D thus appears to be a less efficient enzyme than other known MetAPs in vitro. At saturating substrate concentrations, a plot of Vmax 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 K0.5 of 0.40 microM. Similarly, E. coli MetAP shows n = 2.1 and K0.5 = 0.2 microM. 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 MetAP1 yielded a Hill coefficient (n) of 2.9 and a K0.5 of 0.26 microM for activation by Co2+, as well as a kcat of 25.5 min-1 and a Km of 740 microM for the substrate Met-Pro-p-nitroanilide. The H212A mutation decreased n to 2.2, decreased kcat 60-fold to 0.42 min-1, and increased K0.5 6.5-fold to 1.8 microM. The H212K mutation further decreased n to 1.4, decreased kcat 1800-fold to 0.014 min-1, and increased K0.5 158-fold to 41 microM. 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 Co2+ ion, as indicated by the decreases in n and the increases in K0.5 of the remaining two Co2+ ions, but did not affect the Km 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.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Nov
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
6
pubmed:volume
46
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
12833-43
pubmed:meshHeading
pubmed:year
2007
pubmed:articleTitle
Kinetic and mutational studies of the number of interacting divalent cations required by bacterial and human methionine aminopeptidases.
pubmed:affiliation
Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205, USA.
pubmed:publicationType
Journal Article, Comparative Study, Research Support, U.S. Gov't, Non-P.H.S., Research Support, Non-U.S. Gov't, Research Support, N.I.H., Extramural