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Dual divalent cation requirement of the MutT dGTPase. Kinetic and magnetic resonance studies of the metal and substrate complexes.
J Biol Chem. 1994 Jan 21; 269(3):1794-803.JB

Abstract

Kinetic analyses of both the Mn(2+)- and Mg(2+)-activated hydrolysis of dGTP by MutT show the requirement for two divalent cations. Whereas Mn2+ supports a 20-fold lower kcat (0.19 s-1) than Mg2+ (4.0 s-1), the Km of Mn2+.dGTP (6.3 microM) is 45-fold lower than that of Mg2+.dGTP (284 microM). Adenosine 5'-(alpha,beta-methylenetriphosphate) (AMPCPP) is a linear competitive inhibitor with respect to dGTP with a Ki for Mg2+.AMPCPP (42 microM) which is 57-fold lower than the Ki of Mg2+.AMPCPP (2.4 mM). Such tightening suggests that a metal-bridge E.M2+.NTP.M2+ complex is the catalytically active species. The 12 dissociation constants describing the quaternary MutT.M2+.AMPCPP.M2+ complex were evaluated for both Mn2+ and Mg2+, using EPR and NMR methods. MutT binds a single Mn2+ with a Kd of 130 +/- 40 microM in reasonable agreement with the kinetically determined activator constant of Mn2+ of 230 +/- 72 microM. The MutT.AMPCPP complex binds two Mn2+ ions, the weaker of which has a Kd of 16 +/- 2 microM in agreement with the kinetically determined KmMn2+ of 26 +/- 10 microM. MutT.Mn2+ binds Mn2+.AMPCPP with Kd of 16 +/- 4 microM, whereas MutT alone binds Mn2+.AMPCPP with a Kd of 135 +/- 30 microM. The 17-fold enhanced paramagnetic effect of Mn2+ on the longitudinal relaxation rate of water protons found with the binary MutT.Mn2+ complex decreases to 4.7-fold upon binding of AMPCPP and to 8.7-fold upon binding of Mn2+.AMPCPP, further supporting a metal-bridge MutT.M2+.NTP.M2+ complex. By competition with Mn2+ MutT binds Mg2+ at one site with a Kd of 7.5 mM, and MutT.AMPCPP binds Mg2+ at two sites, the weaker of which has a Kd of 0.9 mM. These values are comparable to the kinetically determined KaMg of 15 +/- 7 mM and KmMg of 1.7 +/- 0.7 mM, respectively. Studies with the racemic, substitution-inert beta, gamma-bidentate tetraamminecobalt (III)-beta,gamma-phosphate-ATP (Co3+(NH3)4ATP) complex show that MutT slowly hydrolyzes only the lambda stereoisomer but requires Mg2+ or Mn2+ to do so, confirming a dual metal ion requirement.

Authors+Show Affiliations

Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

8294428

Citation

Frick, D N., et al. "Dual Divalent Cation Requirement of the MutT dGTPase. Kinetic and Magnetic Resonance Studies of the Metal and Substrate Complexes." The Journal of Biological Chemistry, vol. 269, no. 3, 1994, pp. 1794-803.
Frick DN, Weber DJ, Gillespie JR, et al. Dual divalent cation requirement of the MutT dGTPase. Kinetic and magnetic resonance studies of the metal and substrate complexes. J Biol Chem. 1994;269(3):1794-803.
Frick, D. N., Weber, D. J., Gillespie, J. R., Bessman, M. J., & Mildvan, A. S. (1994). Dual divalent cation requirement of the MutT dGTPase. Kinetic and magnetic resonance studies of the metal and substrate complexes. The Journal of Biological Chemistry, 269(3), 1794-803.
Frick DN, et al. Dual Divalent Cation Requirement of the MutT dGTPase. Kinetic and Magnetic Resonance Studies of the Metal and Substrate Complexes. J Biol Chem. 1994 Jan 21;269(3):1794-803. PubMed PMID: 8294428.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Dual divalent cation requirement of the MutT dGTPase. Kinetic and magnetic resonance studies of the metal and substrate complexes. AU - Frick,D N, AU - Weber,D J, AU - Gillespie,J R, AU - Bessman,M J, AU - Mildvan,A S, PY - 1994/1/21/pubmed PY - 1994/1/21/medline PY - 1994/1/21/entrez SP - 1794 EP - 803 JF - The Journal of biological chemistry JO - J. Biol. Chem. VL - 269 IS - 3 N2 - Kinetic analyses of both the Mn(2+)- and Mg(2+)-activated hydrolysis of dGTP by MutT show the requirement for two divalent cations. Whereas Mn2+ supports a 20-fold lower kcat (0.19 s-1) than Mg2+ (4.0 s-1), the Km of Mn2+.dGTP (6.3 microM) is 45-fold lower than that of Mg2+.dGTP (284 microM). Adenosine 5'-(alpha,beta-methylenetriphosphate) (AMPCPP) is a linear competitive inhibitor with respect to dGTP with a Ki for Mg2+.AMPCPP (42 microM) which is 57-fold lower than the Ki of Mg2+.AMPCPP (2.4 mM). Such tightening suggests that a metal-bridge E.M2+.NTP.M2+ complex is the catalytically active species. The 12 dissociation constants describing the quaternary MutT.M2+.AMPCPP.M2+ complex were evaluated for both Mn2+ and Mg2+, using EPR and NMR methods. MutT binds a single Mn2+ with a Kd of 130 +/- 40 microM in reasonable agreement with the kinetically determined activator constant of Mn2+ of 230 +/- 72 microM. The MutT.AMPCPP complex binds two Mn2+ ions, the weaker of which has a Kd of 16 +/- 2 microM in agreement with the kinetically determined KmMn2+ of 26 +/- 10 microM. MutT.Mn2+ binds Mn2+.AMPCPP with Kd of 16 +/- 4 microM, whereas MutT alone binds Mn2+.AMPCPP with a Kd of 135 +/- 30 microM. The 17-fold enhanced paramagnetic effect of Mn2+ on the longitudinal relaxation rate of water protons found with the binary MutT.Mn2+ complex decreases to 4.7-fold upon binding of AMPCPP and to 8.7-fold upon binding of Mn2+.AMPCPP, further supporting a metal-bridge MutT.M2+.NTP.M2+ complex. By competition with Mn2+ MutT binds Mg2+ at one site with a Kd of 7.5 mM, and MutT.AMPCPP binds Mg2+ at two sites, the weaker of which has a Kd of 0.9 mM. These values are comparable to the kinetically determined KaMg of 15 +/- 7 mM and KmMg of 1.7 +/- 0.7 mM, respectively. Studies with the racemic, substitution-inert beta, gamma-bidentate tetraamminecobalt (III)-beta,gamma-phosphate-ATP (Co3+(NH3)4ATP) complex show that MutT slowly hydrolyzes only the lambda stereoisomer but requires Mg2+ or Mn2+ to do so, confirming a dual metal ion requirement. SN - 0021-9258 UR - https://www.unboundmedicine.com/medline/citation/8294428/Dual_divalent_cation_requirement_of_the_MutT_dGTPase__Kinetic_and_magnetic_resonance_studies_of_the_metal_and_substrate_complexes_ L2 - http://www.jbc.org/cgi/pmidlookup?view=long&pmid=8294428 DB - PRIME DP - Unbound Medicine ER -