Mechanism of adenylate kinase. 1. Use of oxygen-17 NMR to study the binding properties of substrates

Wisner, Daniel A.; Steginsky, C A; Shyy, Yeun Jund; Tsai, Ming‐Daw

doi:10.1021/ja00295a048

Cited by 11 publications

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“…Previous 170 NMR studies completed in our laboratory (Wisner et al, 1985) by using -, ß-, and y-170-labeled ATP with AK suggest qualitatively that the terminal phosphate possesses more mobility than the ß phosphate which, in turn, possesses more mobility than the a phosphate for the AK-ATP complex. Although use of ATP would be preferable to AMPPCP, deuterium NMR is much more quantitative than 170 NMR since I70 (I = 5/2) has a large quadrupole moment and the relaxation behavior becomes multiexponential outside the extreme narrowing region (Forsen et al, 1987).…”

Section: Concentration Of Amp (M)mentioning

confidence: 88%

Mechanism of adenylate kinase. Is there a relationship between local substrate dynamics, and local binding energy, and the catalytic mechanism?

Sanders¹,

Tian²,

Tsai³

1989

Biochemistry

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Adenylyl (beta,gamma-methylene)diphosphonic acid (AMPPCP) labeled with deuterium at the adenine ring ([8-2H]AMPPCP) and at the beta,gamma-methylene group (AMPPCD2P), as well as adenosine 5'-monophosphate labeled at the adenine ring ([8-2H]AMP), was synthesized and used for deuterium nuclear magnetic resonance (NMR) determination of effective correlation times (tau c) of the free nucleotide and the complexes with adenylate kinase (AK). Extensive and rigorous control experiments and theoretical analysis were performed to justify the validity of the experimental approaches, particularly the fast exchange condition, and the reliability of the tau c values obtained. For the free nucleotide, the results suggest that the phosphonate group of free AMPPCP possesses appreciable local mobility relative to the adenine ring and that complexation with Mg2+ greatly reduced such a local mobility. For the complexes with AK, effective tau c values of 7, 15, 28, 28, and 27 ns were obtained for AMPPCD2P, MgAMPPCD2P, [8-2H]AMPPCP, Mg[8-2H]AMPPCP, and [8-2H]AMP, respectively. These results suggest that the adenine ring of substrates is rigidly bound in all cases, that the phosphonate chain of AMPPCP possesses considerable local mobility, and that Mg2+ reduces such local mobility but does not totally immobilize it. The local dynamics of the analogues bound to AK was correlated with local binding energies for the binding of MgAMPPCP and MgATP to AK estimated from the binding studies by proton NMR and other techniques, in conjunction with the binding theory of Jencks [Jencks, W. P. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 4046-4050]. The results suggest that no general correlation exists between the local rigidity of portions of a bound substrate and the corresponding (ground state) local binding energy contributed by these portions. In particular, the adenosine moiety contributes little to the binding energy despite the fact that the adenine ring is rigidly bound; the triphosphate (PPPi) moiety behaves oppositely; Mg2+ immobilizes the triphosphate chain but does not enhance binding. Finally, isomers of the substitution-inert beta,gamma-bidentate Cr(III) complexes of adenosine 5'-triphosphate (CrATP) were used to probe two unresolved catalytic problems implicitly related to the local mobility of the phosphonate chain of AMPPCP in the AK-MgAMPPCP complex. The first problem concerns the result of electron paramagnetic resonance (EPR) studies that (Rp)- but not (Sp)-[beta-17O]ATP caused a line broadening in the Mn(II) EPR spectrum of the AK-MnATP complex [Kalbitzer, H. R., Marquetant, R., Connolly, B. A., & Goody, R. S. (1983) Eur. J. Biochem. 133, 221-227].(ABSTRACT TRUNCATED AT 400 WORDS)

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Section: Concentration Of Amp (M)mentioning

confidence: 88%

Mechanism of adenylate kinase. Is there a relationship between local substrate dynamics, and local binding energy, and the catalytic mechanism?

Sanders¹,

Tian²,

Tsai³

1989

Biochemistry

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“…A p p l i c a t i o n o f s o l u t i o n 1 7 O N M R t o t h e s t u d y o f e n z y m e -s u b s t r a t e complexes was demonstrated some time ago (Wisner et al 1985). However, 17 O NMR spectra of enzyme-substrate complexes are often plagued by broad lines resulting from efficient quadrupole relaxation, especially when t h e ligand is tightly held.…”

Section: Binding Of Small Ligands To Biomoleculesmentioning

confidence: 99%

Recent developments in solid-state nuclear magnetic resonance of quadrupolar nuclei and applications to biological systems

Wu¹

1998

Biochem. Cell Biol.

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Recent advances in nuclear magnetic resonance (NMR) methodology and improvements in high-field NMR instrumentation have generated a new wave of research interests in the application of solid-state NMR to the study of quadrupolar nuclei. These developments now permit increasingly complex biological systems to be probed by quadrupolar NMR. In this review I describe a few recent developments in NMR studies of quadrupolar nuclei and demonstrate the potential of solid-state quadrupolar NMR in the study of biological systems. In particular, I discuss the application of solid-state NMR of (17)O, 67Zn, 59Co, 23Na, and 39K nuclei with a prognosis for future work.

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“…This is paper 2 in the series "Mechanism of Adenylate Kinase". For paper 1, see Wisner et al (1985).…”

Section: Furthermentioning

confidence: 99%

“…Although uncomplexed ATP is not a natural substrate of AK, the issue of whether it binds to the AMP site is important in understanding the active-site structure of AK and in properly interpreting the results of biochemical and physical studies. For example, the binding properties of uncomplexed ATP to AK have been characterized by NMR (McDonald et al, 1975), 31P NMR (Nageswara et al, 1978), 170 NMR (Wisner et al, 1985), UV difference spectroscopy (Hamada et al, 1979), and CD difference spectroscopy (Yazawa & Noda, 1976). These results would require reinterpretation if ATP can bind to both sites, or even to the AMP site exclusively, in the absence of Mg2+.…”

Section: Furthermentioning

confidence: 99%

Mechanism of adenylate kinase. Does adenosine 5'-triphosphate bind to the adenosine 5'-monophosphate site?

Shyy¹,

Tian²,

Tsai³

1987

Biochemistry

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Although the substrate binding properties of adenylate kinase (AK) have been studied extensively by various biochemical and biophysical techniques, it remains controversial whether uncomplexed adenosine 5'-triphosphate (ATP) binds to the adenosine 5'-monophosphate (AMP) site of AK. We present two sets of experiments which argue against binding of ATP to the AMP site. (a) 31P nuclear magnetic resonance titration of ATP with AK indicated a 1:1 stoichiometry on the basis of changes in coupling constants and line widths. This ruled out binding of ATP to both sites. (b) ATP and MgATP were found to behave similarly by protecting AK from spontaneous inactivation while AMP showed only a small degree of protection. Such inactivation could also be protected or reversed by dithioerythritol and is most likely due to oxidation of sulfhydryl groups, one of which (cysteine-25) is located near the MgATP site. The results support binding of ATP to the MgATP site predominantly, instead of the AMP site, in the absence of Mg2+.

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Mechanism of adenylate kinase. 1. Use of oxygen-17 NMR to study the binding properties of substrates

Cited by 11 publications

References 0 publications

Mechanism of adenylate kinase. Is there a relationship between local substrate dynamics, and local binding energy, and the catalytic mechanism?

Mechanism of adenylate kinase. Is there a relationship between local substrate dynamics, and local binding energy, and the catalytic mechanism?

Recent developments in solid-state nuclear magnetic resonance of quadrupolar nuclei and applications to biological systems

Mechanism of adenylate kinase. Does adenosine 5'-triphosphate bind to the adenosine 5'-monophosphate site?

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