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25Mg NMR studies of yeast enolase and rabbit muscle pyruvate kinase.
Arch Biochem Biophys. 1992 Mar; 293(2):264-73.AB

Abstract

25Mg NMR spectroscopy was used to study the interactions of the activating cations with their respective binding sites in the enzymes yeast enolase and rabbit muscle pyruvate kinase (PK). Titration of Mg2+ with enolase allows for the calculation of 1/T2 for Mg2+ bound at site I of 1510 s-1 and a quadrupolar coupling constant chi = 0.30 MHz. Titration of Mg2+ with enolase in the presence of 2-phosphoglycerate (PGA) and Zn2+, where Zn2+ binds specifically at site I, gives a 1/T2 for Mg2+ bound at site II of 4000 s-1 (chi = 0.49 MHz). The Mg2+ at site II appears to be more anisotropic than Mg2+ at site I. The titration of site I of the enolase-Mg-PGA-Mg complex with Zn2+ or Mn2+ shows a simple displacement of the Mg2+. No paramagnetic effects by Mn2+ on 25Mg relaxation were observed. Temperature studies of the 25Mg resonance show that fast exchange of the Mg2+ occurs under these conditions. From the lack of a paramagnetic effect, the distance between the cations at sites I and II must be more than 6-9 A. This distance limits the location, hence the function, of the cation at site II for catalytic activity. Titration of Mg2+ with PK gives a 1/T2 for bound Mg2+ of 2200 s-1 (chi = 0.24 MHz). A titration of Mg2+ with PK in the presence of the inhibitor oxalate gives a 1/T2 of 400 s-1. The temperature dependence of 25Mg relaxation in the PK-Mg-oxalate complex is consistent with slow exchange (Ea = 6.1 +/- 1.6 kcal/mol). The enzyme-bound cation is more tightly sequestered by the addition of a ligand that binds directly to the cation. An investigation of the 25Mg relaxation in the PK-Mn-oxalate-Mg-ATP complex, where the Mg2+ is bound to the nucleotide and the Mn2+ was enzyme bound, was not successful due to precipitation of PK under experimental conditions and the short T2 relaxation for 25Mg in this complex. The applications of 25Mg NMR have been useful in partially describing the properties of the bound Mg2+ in these two metal-requiring enzymes.

Authors+Show Affiliations

Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556.No affiliation info available

Pub Type(s)

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

Language

eng

PubMed ID

1311162

Citation

Lee, M E., and T Nowak. "25Mg NMR Studies of Yeast Enolase and Rabbit Muscle Pyruvate Kinase." Archives of Biochemistry and Biophysics, vol. 293, no. 2, 1992, pp. 264-73.
Lee ME, Nowak T. 25Mg NMR studies of yeast enolase and rabbit muscle pyruvate kinase. Arch Biochem Biophys. 1992;293(2):264-73.
Lee, M. E., & Nowak, T. (1992). 25Mg NMR studies of yeast enolase and rabbit muscle pyruvate kinase. Archives of Biochemistry and Biophysics, 293(2), 264-73.
Lee ME, Nowak T. 25Mg NMR Studies of Yeast Enolase and Rabbit Muscle Pyruvate Kinase. Arch Biochem Biophys. 1992;293(2):264-73. PubMed PMID: 1311162.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - 25Mg NMR studies of yeast enolase and rabbit muscle pyruvate kinase. AU - Lee,M E, AU - Nowak,T, PY - 1992/3/1/pubmed PY - 1992/3/1/medline PY - 1992/3/1/entrez SP - 264 EP - 73 JF - Archives of biochemistry and biophysics JO - Arch. Biochem. Biophys. VL - 293 IS - 2 N2 - 25Mg NMR spectroscopy was used to study the interactions of the activating cations with their respective binding sites in the enzymes yeast enolase and rabbit muscle pyruvate kinase (PK). Titration of Mg2+ with enolase allows for the calculation of 1/T2 for Mg2+ bound at site I of 1510 s-1 and a quadrupolar coupling constant chi = 0.30 MHz. Titration of Mg2+ with enolase in the presence of 2-phosphoglycerate (PGA) and Zn2+, where Zn2+ binds specifically at site I, gives a 1/T2 for Mg2+ bound at site II of 4000 s-1 (chi = 0.49 MHz). The Mg2+ at site II appears to be more anisotropic than Mg2+ at site I. The titration of site I of the enolase-Mg-PGA-Mg complex with Zn2+ or Mn2+ shows a simple displacement of the Mg2+. No paramagnetic effects by Mn2+ on 25Mg relaxation were observed. Temperature studies of the 25Mg resonance show that fast exchange of the Mg2+ occurs under these conditions. From the lack of a paramagnetic effect, the distance between the cations at sites I and II must be more than 6-9 A. This distance limits the location, hence the function, of the cation at site II for catalytic activity. Titration of Mg2+ with PK gives a 1/T2 for bound Mg2+ of 2200 s-1 (chi = 0.24 MHz). A titration of Mg2+ with PK in the presence of the inhibitor oxalate gives a 1/T2 of 400 s-1. The temperature dependence of 25Mg relaxation in the PK-Mg-oxalate complex is consistent with slow exchange (Ea = 6.1 +/- 1.6 kcal/mol). The enzyme-bound cation is more tightly sequestered by the addition of a ligand that binds directly to the cation. An investigation of the 25Mg relaxation in the PK-Mn-oxalate-Mg-ATP complex, where the Mg2+ is bound to the nucleotide and the Mn2+ was enzyme bound, was not successful due to precipitation of PK under experimental conditions and the short T2 relaxation for 25Mg in this complex. The applications of 25Mg NMR have been useful in partially describing the properties of the bound Mg2+ in these two metal-requiring enzymes. SN - 0003-9861 UR - https://www.unboundmedicine.com/medline/citation/1311162/25Mg_NMR_studies_of_yeast_enolase_and_rabbit_muscle_pyruvate_kinase_ L2 - https://linkinghub.elsevier.com/retrieve/pii/0003-9861(92)90394-C DB - PRIME DP - Unbound Medicine ER -