A number of health problems, such as muscle and metabolic diseases, are related to the role of ATP-utilizing enzymes which play a key role in a variety of biochemical pathways. All these enzymes require Mg(II) as an obligatory component of their reactions. Understanding the catalytic activity of the enzymes on a molecular basis requires knowledge of the structures of the reactants and products bound at the active sites of the enzymes. The goal of this project is to establish the active-site structures of three classes of ATP-utilizing enzymes viz. phosphoryl transfer, nucleotidyl transfer and pyrophosphoryl transfer by using NMR spectroscopy which is one of the few techniques available for structural investigations in liquids. Two types of experiments will be performed to obtain structural data: (i) spin-lattice relaxation measurements of 31P and selectively labelled 13C and 15N nuclei of the substrates in their enzyme-bound complexes in the presence of such activating paramagnetic cations as Mn(II) and Co(II) substituted for the normal activator Mg(II) and (ii) homonuclear proton NOE measurements on nucleotides in their enzyme complexes. The first method yields distances from the cation to the 31P, 13C, and 15N nuclei in the substrates and the second method provides information on the interproton distances on the nucleotides. Measurements will also be made using thionucleotide analogs, in place of normal nucleotides, in order to introduce stereochemical information into the structure data. The proposed measurements represent the maximal distance data that NMR methodology can provide on the substrates bound to the enzymes chosen. A special effort will be made to assess an acceptable range for each distance obtained from these studies as implied by the measurements themselves and the theoretical basis of calculating these distances from the data. Active-site structures compatible with the distance data acquired as above, will be constructed using appropriate iterative procedures.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM043966-09
Application #
6125432
Study Section
Biochemistry Study Section (BIO)
Program Officer
Wehrle, Janna P
Project Start
1989-07-01
Project End
2002-11-30
Budget Start
1999-12-01
Budget End
2002-11-30
Support Year
9
Fiscal Year
2000
Total Cost
$221,101
Indirect Cost
Name
Indiana University-Purdue University at Indianapolis
Department
Physics
Type
Schools of Arts and Sciences
DUNS #
005436803
City
Indianapolis
State
IN
Country
United States
Zip Code
46202
Ray, Bruce D; Scott, Joshua; Yan, Honggao et al. (2009) Productive versus unproductive nucleotide binding in yeast guanylate kinase mutants: comparison of R41M with K14M by proton two dimensional transferred NOESY. Biochemistry 48:5532-40
Ray, Bruce D; Jarori, Gotam K; Raghunathan, Vidya et al. (2005) Conformations of nucleotides bound to wild type and Y78F mutant yeast guanylate kinase: proton two-dimensional transferred NOESY measurements. Biochemistry 44:13762-70
Ray, Bruce D; Khoroshev, Mikhail I; Ue, Kathleen et al. (2002) Changes in the 31P NMR spectrum of rabbit muscle myosin subfragment 1. MgADP with temperature. Arch Biochem Biophys 402:243-8
Ray, Bruce D; Jarori, Gotam K; Rao, B D Nageswara (2002) Quantitation of movement of the phosphoryl group during catalytic transfer in the arginine kinase reaction: 31P relaxation measurements on enzyme-bound equilibrium mixtures. J Biomol NMR 23:13-21
Lin, Y; Nageswara Rao, B D (2000) Structural characterization of adenine nucleotides bound to Escherichia coli adenylate kinase. 2. 31P and 13C relaxation measurements in the presence of cobalt(II) and manganese(II). Biochemistry 39:3647-55
Lin, Y; Nageswara Rao, B D (2000) Structural characterization of adenine nucleotides bound to Escherichia coli adenylate kinase. 1. Adenosine conformations by proton two-dimensional transferred nuclear Overhauser effect spectroscopy. Biochemistry 39:3636-46
Ray, B D; Jarori, G K; Nageswara Rao, B D (1999) Paramagnetic effects on nuclear relaxation in enzyme-bound Co(II)-adenine nucleotide complexes: relative contributions of dipolar and scalar interactions. J Magn Reson 136:130-3
Raghunathan, V; Chau, M H; Ray, B D et al. (1999) Structural characterization of manganese(II)-nucleotide complexes bound to yeast 3-phosphoglycerate kinase: 13C relaxation measurements using [U-13C]ATP and [U-13C]ADP. Biochemistry 38:15597-605
Murali, N; Lin, Y; Mechulam, Y et al. (1997) Adenosine conformations of nucleotides bound to methionyl tRNA synthetase by transferred nuclear Overhauser effect spectroscopy. Biophys J 72:2275-84
Ray, B D; Chau, M H; Fife, W K et al. (1996) Conformation of manganese(II)-nucleotide complexes bound to rabbit muscle creatine kinase: 13C NMR measurements using [2-13C]ATP and [2-13C]ADP. Biochemistry 35:7239-46

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