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

Xiaoyi V. Hu, Xiaochun Chen, Chung Han Kee, Albert S. Mildvan, Jun Liu

Research output: Contribution to journalArticle

Abstract

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 MetAPID of 0.39 min-1 is 280-fold lower, and the Km 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 K0.5 of 0.40 μM. Similarly, E. coli MetAP shows n = 2.1 and K0.5 = 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 K0.5 of 0.26 μM for activation by Co2+, as well as a kcat of 25.5 min -1 and a Km 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 K0.5 6.5-fold to 1.8 μM. The H212K mutation further decreased n to 1.4, decreased kcat 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 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.

Original languageEnglish (US)
Pages (from-to)12833-12843
Number of pages11
JournalBiochemistry®
Volume46
Issue number44
DOIs
StatePublished - Nov 6 2007

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Aminopeptidases
Divalent Cations
Methionine
Kinetics
Ions
Escherichia coli
Catalysis
Metals
Mutation
Biochemistry
Substrates
Chemical activation
X-Rays
X rays
Enzymes
Post Translational Protein Processing
Kinetic parameters
Metal ions
Catalytic Domain
Protein Isoforms

ASJC Scopus subject areas

  • Biochemistry

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Kinetic and mutational studies of the number of interacting divalent cations required by bacterial and human methionine aminopeptidases. / Hu, Xiaoyi V.; Chen, Xiaochun; Kee, Chung Han; Mildvan, Albert S.; Liu, Jun.

In: Biochemistry®, Vol. 46, No. 44, 06.11.2007, p. 12833-12843.

Research output: Contribution to journalArticle

Hu, Xiaoyi V. ; Chen, Xiaochun ; Kee, Chung Han ; Mildvan, Albert S. ; Liu, Jun. / Kinetic and mutational studies of the number of interacting divalent cations required by bacterial and human methionine aminopeptidases. In: Biochemistry®. 2007 ; Vol. 46, No. 44. pp. 12833-12843.
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title = "Kinetic and mutational studies of the number of interacting divalent cations required by bacterial and human methionine aminopeptidases",
abstract = "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 MetAPID of 0.39 min-1 is 280-fold lower, and the Km 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 K0.5 of 0.40 μM. Similarly, E. coli MetAP shows n = 2.1 and K0.5 = 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 K0.5 of 0.26 μM for activation by Co2+, as well as a kcat of 25.5 min -1 and a Km 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 K0.5 6.5-fold to 1.8 μM. The H212K mutation further decreased n to 1.4, decreased kcat 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 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.",
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TY - JOUR

T1 - Kinetic and mutational studies of the number of interacting divalent cations required by bacterial and human methionine aminopeptidases

AU - Hu, Xiaoyi V.

AU - Chen, Xiaochun

AU - Kee, Chung Han

AU - Mildvan, Albert S.

AU - Liu, Jun

PY - 2007/11/6

Y1 - 2007/11/6

N2 - 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 MetAPID of 0.39 min-1 is 280-fold lower, and the Km 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 K0.5 of 0.40 μM. Similarly, E. coli MetAP shows n = 2.1 and K0.5 = 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 K0.5 of 0.26 μM for activation by Co2+, as well as a kcat of 25.5 min -1 and a Km 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 K0.5 6.5-fold to 1.8 μM. The H212K mutation further decreased n to 1.4, decreased kcat 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 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.

AB - 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 MetAPID of 0.39 min-1 is 280-fold lower, and the Km 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 K0.5 of 0.40 μM. Similarly, E. coli MetAP shows n = 2.1 and K0.5 = 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 K0.5 of 0.26 μM for activation by Co2+, as well as a kcat of 25.5 min -1 and a Km 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 K0.5 6.5-fold to 1.8 μM. The H212K mutation further decreased n to 1.4, decreased kcat 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 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.

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