Analysis of Equilibrium Data from Proton Magnetic Relaxation Rates of Water for Manganese-Nucleotide-Kinase Ternary Complexes

Reed, George H.; Cohn, Mildred; O’Sullivan, W.J.

doi:10.1016/s0021-9258(18)62568-2

Cited by 109 publications

(45 citation statements)

References 14 publications

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“…This plot clearly shows that the NOE is practically unchanged for ADP concentrations up to ~6 mM and then starts to increase rapidly with increasing ligand concentration, going up by a factor of nearly 5 for 10 mM ADP. Since the Hl,-H2/ distance is considered to be invariant in any conformation of adenosine (DeLeeuw et al, 1980;Rosevear et al, 1983), the increase in NOE at high concentrations must arise from an increase in p* because r£ is not expected to change with concentrations in this range.2 On the basis of the known dissociation constants (Reed et al, 1970), the occupation of the active site changes at the most from 95% to 99% with the increase in concentrations. The increase in by nearly a factor of 5 must, therefore, be due to adventitious binding of the nucleotide to the enzyme.…”

Section: Results and Analysismentioning

confidence: 99%

Two-dimensional transferred nuclear Overhauser effect spectroscopy (TRNOESY) studies of nucleotide conformations in creatine kinase complexes: Effects due to weak nonspecific binding

Murali

Jarori²,

Landy³

et al. 1993

Biochemistry

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The conformations of the adenosine moiety of MgADP and MgATP bound to rabbit muscle creatine kinase were investigated by two-dimensional transferred nuclear Overhauser effect spectroscopy (TRNOESY). The effects arising from adventitious binding of the ligands to the enzyme on the measurements were delineated. It was shown that, with sample protocols typically used thus far with the TRNOE method (enzyme, approximately 1 mM; ligand, approximately 10 mM), the TRNOESY pattern for the nucleotides with creatine kinase is similar to that with gamma-globulin and bovine serum albumin, which do not have specific nucleotide binding site(s). Measurements of NOE between the H1'-H2' proton pair as a function of ligand concentrations with the enzyme-ligand ratio kept constant at 1:10 showed that, for ligand concentrations over about 3-4 mM, weak nonspecific binding makes a significant contribution to the observed NOE. Thus the NOE values relevant for the determination of the nucleotide conformation at the active site were measured at nucleotide concentrations of about 1.5 mM. The TRNOE buildup curves for all the ligand-proton pairs were analyzed using a complete relaxation matrix approach. The interproton distances derived from the NOE's were then used as constraints in elucidating the ligand structure by using the program CHARMm. The NOE-determined structures of both MgADP and MgATP bound to creatine kinase correspond to an anti conformation with the glycosidic angle (O'4-C'1-N9-C8) chi = 51 +/- 5 degrees. The ribose pucker nominally representative of these data is a O4'T with a phase angle of pseudorotation (p) of 70.5 degrees.

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Section: Results and Analysismentioning

confidence: 99%

Two-dimensional transferred nuclear Overhauser effect spectroscopy (TRNOESY) studies of nucleotide conformations in creatine kinase complexes: Effects due to weak nonspecific binding

Murali

Jarori²,

Landy³

et al. 1993

Biochemistry

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“…concentrations it exhibits almost a linear increase. The smallest ATP concentration (1.0 mM) used here was large enough to saturate the active sites (0.1 mM) of pyruvate kinase in excess of 90% (K¿ = 0.022 mM, Reed et al (1970)). The large linear increase in NOE is clearly not due to the small increase of 10% in the saturation fraction at the active site.…”

Section: Resultsmentioning

confidence: 99%

Two-Dimensional Transferred Nuclear Overhauser Effect Spectroscopy Study of the Conformation of MgATP Bound at the Active and Ancillary Sites of Rabbit Muscle Pyruvate Kinase

Jarori

Murali²,

Rao³

1994

Biochemistry

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Pyruvate kinase binds one adenosine 5'-triphosphate (ATP) molecule at its active site and another at an ancillary site on each subunit. In order to determine the conformation of ATP bound at these sites, proton transferred two-dimensional nuclear Overhauser effect spectroscopy (TRNOESY) measurements were made at 500 MHz and 10 degrees C for several mixing times in the range 40-200 ms. The NOE values for the proton pair H1'-H2' of ribose (which are 2.9 +/- 0.2 A apart, irrespective of nucleotide conformation) as a function of ligand concentration (1-10 mM ATP), with the ratio of ligand to enzyme being kept constant, indicate that at higher ligand concentrations adventitious binding of ATP at nonspecific site(s) makes a major contribution to the observed NOEs. When the ligand concentration is < 2 mM, site-specific NOEs can be measured. Furthermore, addition of phosphoenolpyruvate (PEP) to the enzyme-MgATP sample results in competitive displacement of MgATP from the active site and reduces the observed NOE to that arising exclusively at the ancillary site, thus allowing the measurement of site-specific NOEs. The interproton distances determined from such site-specific NOE buildup curves were used as constraints in CHARMm to obtain the structure of MgATP. At the active site, MgATP has a glycosidic torsion chi = 44 +/- 5 degrees and the phase angle of pseudorotation for ribose P = 42.4 degrees. At the ancillary site chi = 46 +/- 5 degrees and P = 127.6 degrees. Thus the orientation of the adenine with respect to the sugar moiety is the same at both sites.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…The concentrations chosen for the measurements presented here may be seen in Table 1. On the basis of the known dissociation constants for creatine kinase complexes (Reed et al, 1970), the fractional concentration of the paramagnetic complex free in solution ([M‚S]/[E‚M‚S]) never exceeds ∼3%.…”

Section: Methodsmentioning

confidence: 99%

Conformation of Manganese(II)−Nucleotide Complexes Bound to Rabbit Muscle Creatine Kinase: ¹³C NMR Measurements Using [2-¹³C]ATP and [2-¹³C]ADP

et al. 1996

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Conformations of cation-nucleotide complexes bound to rabbit muscle creatine kinase were investigated by measuring paramagnetic effects on 13C spin relaxation in E.Mn[2-13C]ATP and E.Mn[2-13C]ADP at three different frequencies, viz., 50, 75, and 125 MHz, and as a function of temperature in the range of 7-35 degrees C (at 75 MHz). Arrhenius plots of the temperature dependencies of relaxation rates show a positive slope with low activation energies of 1.3 +/- 0.2 kcal/mol and 2.0 +/- 0.2 kcal/mol for E.Mn ATP and E.MnADP, respectively. The relaxation rates of both complexes show strong frequency dependence, indicating that these rates are not exchange limited. Analysis of the data yields Mn(II)-2C distances of 10.0 +/- 0.5 A for E.MnATP and 8.6 +/- 0.5 A for E.MnADP. These data were interpreted, along with previously published information, on the location of the cation with respect to the phosphate chain [Jarori, G. K., Ray, B.D., & Nageswara Rao, B. D. (1985) Biochemistry 24, 3487-3494], and on the adenosine conformation [Murali, N., Jarori, G. K., & Nageswara Rao, B. D. (1993) Biochemistry 32, 12941-12948] in these complexes. The Mn(II)-2C distances depend on the orientation of the phosphate chain relative to the adenosine moiety. Conformational searches were performed by varying the two torsion angles, phi 1 (C4'-C5'-O5'-P alpha), and phi 2 (C5'-O5'-P alpha-O alpha beta), along with CHARMm energy computations, in order to determine acceptable conformations compatible with the distances determined. The significant difference in the Mn(II)-2C distances in E.MnATP and E.MnADP is indicative of the structural alterations occurring at the active site as the enzyme turns over.

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Analysis of Equilibrium Data from Proton Magnetic Relaxation Rates of Water for Manganese-Nucleotide-Kinase Ternary Complexes

Cited by 109 publications

References 14 publications

Two-dimensional transferred nuclear Overhauser effect spectroscopy (TRNOESY) studies of nucleotide conformations in creatine kinase complexes: Effects due to weak nonspecific binding

Two-dimensional transferred nuclear Overhauser effect spectroscopy (TRNOESY) studies of nucleotide conformations in creatine kinase complexes: Effects due to weak nonspecific binding

Two-Dimensional Transferred Nuclear Overhauser Effect Spectroscopy Study of the Conformation of MgATP Bound at the Active and Ancillary Sites of Rabbit Muscle Pyruvate Kinase

Conformation of Manganese(II)−Nucleotide Complexes Bound to Rabbit Muscle Creatine Kinase: ¹³C NMR Measurements Using [2-¹³C]ATP and [2-¹³C]ADP

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Analysis of Equilibrium Data from Proton Magnetic Relaxation Rates of Water for Manganese-Nucleotide-Kinase Ternary Complexes

Cited by 109 publications

References 14 publications

Two-dimensional transferred nuclear Overhauser effect spectroscopy (TRNOESY) studies of nucleotide conformations in creatine kinase complexes: Effects due to weak nonspecific binding

Two-dimensional transferred nuclear Overhauser effect spectroscopy (TRNOESY) studies of nucleotide conformations in creatine kinase complexes: Effects due to weak nonspecific binding

Two-Dimensional Transferred Nuclear Overhauser Effect Spectroscopy Study of the Conformation of MgATP Bound at the Active and Ancillary Sites of Rabbit Muscle Pyruvate Kinase

Conformation of Manganese(II)−Nucleotide Complexes Bound to Rabbit Muscle Creatine Kinase: 13C NMR Measurements Using [2-13C]ATP and [2-13C]ADP

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Conformation of Manganese(II)−Nucleotide Complexes Bound to Rabbit Muscle Creatine Kinase: ¹³C NMR Measurements Using [2-¹³C]ATP and [2-¹³C]ADP