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Ternary borate-nucleoside complex stabilization by ribonuclease A demonstrates phosphate mimicry.

ABSTRACT: Phosphate esters play a central role in cellular energetics, biochemical activation, signal transduction and conformational switching. The structural homology of the borate anion with phosphate, combined with its ability to spontaneously esterify hydroxyl groups, suggested that phosphate ester recognition sites on proteins might exhibit significant affinity for nonenzymatically formed borate esters. (11)B NMR studies and activity measurements on ribonuclease A (RNase A) in the presence of borate and several cytidine analogs demonstrate the formation of a stable ternary RNase A.3'-deoxycytidine-2'-borate ternary complex that mimics the complex formed between RNase A and a 2'-cytidine monophosphate (2'-CMP) inhibitor. Alternatively, no slowly exchanging borate resonance is observed for a ternary RNase A, borate, 2'-deoxycytidine mixture, demonstrating the critical importance of the 2'-hydroxyl group for complex formation. Titration of the ternary complex with 2'-CMP shows that it can displace the bound borate ester with a binding constant that is close to the reported inhibition constant of RNase A by 2'-CMP. RNase A binding of a cyclic cytidine-2',3'-borate ester, which is a structural homolog of the cytidine-2',3'-cyclic phosphate substrate, could also be demonstrated. The apparent dissociation constant for the cytidine-2',3'-borate.RNase A complex is 0.8 mM, which compares with a Michaelis constant of 11 mM for cytidine-2',3'-cyclic phosphate at pH 7, indicating considerably stronger binding. However, the value is 1,000-fold larger than the reported dissociation constant of the RNase A complex with uridine-vanadate. These results are consistent with recent reports suggesting that in situ formation of borate esters that mimic the corresponding phosphate esters supports enzyme catalysis.

SUBMITTER: Gabel SA

PROVIDER: S-EPMC2888155 | biostudies-literature | 2008 Feb

REPOSITORIES: biostudies-literature

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Ternary borate-nucleoside complex stabilization by ribonuclease A demonstrates phosphate mimicry.

Gabel Scott A SA London Robert E RE

Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry 20071024 2

Phosphate esters play a central role in cellular energetics, biochemical activation, signal transduction and conformational switching. The structural homology of the borate anion with phosphate, combined with its ability to spontaneously esterify hydroxyl groups, suggested that phosphate ester recognition sites on proteins might exhibit significant affinity for nonenzymatically formed borate esters. (11)B NMR studies and activity measurements on ribonuclease A (RNase A) in the presence of borate ...[more]

PMID: 17957392

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Project description:BACKGROUND: The structure, function and reaction mechanism of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) have been extensively studied. Based on these studies, three anion binding sites have been identified, one 'Ps' site (for binding the C-3 phosphate of the substrate) and two sites, 'Pi' and 'new Pi', for inorganic phosphate. According to the original flip-flop model, the substrate phosphate group switches from the 'Pi' to the 'Ps' site during the multistep reaction. In light of the discovery of the 'new Pi' site, a modified flip-flop mechanism, in which the C-3 phosphate of the substrate binds to the 'new Pi' site and flips to the 'Ps' site before the hydride transfer, was proposed. An alternative model based on a number of structures of B. stearothermophilus GAPDH ternary complexes (non-covalent and thioacyl intermediate) proposes that in the ternary Michaelis complex the C-3 phosphate binds to the 'Ps' site and flips from the 'Ps' to the 'new Pi' site during or after the redox step. RESULTS: We determined the crystal structure of Cryptosporidium parvum GAPDH in the apo and holo (enzyme + NAD) state and the structure of the ternary enzyme-cofactor-substrate complex using an active site mutant enzyme. The C. parvum GAPDH complex was prepared by pre-incubating the enzyme with substrate and cofactor, thereby allowing free movement of the protein structure and substrate molecules during their initial encounter. Sulfate and phosphate ions were excluded from purification and crystallization steps. The quality of the electron density map at 2A resolution allowed unambiguous positioning of the substrate. In three subunits of the homotetramer the C-3 phosphate group of the non-covalently bound substrate is in the 'new Pi' site. A concomitant movement of the phosphate binding loop is observed in these three subunits. In the fourth subunit the C-3 phosphate occupies an unexpected site not seen before and the phosphate binding loop remains in the substrate-free conformation. Orientation of the substrate with respect to the active site histidine and serine (in the mutant enzyme) also varies in different subunits. CONCLUSION: The structures of the C. parvum GAPDH ternary complex and other GAPDH complexes demonstrate the plasticity of the substrate binding site. We propose that the active site of GAPDH can accommodate the substrate in multiple conformations at multiple locations during the initial encounter. However, the C-3 phosphate group clearly prefers the 'new Pi' site for initial binding in the active site.

Dataset Information

Ternary borate-nucleoside complex stabilization by ribonuclease A demonstrates phosphate mimicry.

Publications

Ternary borate-nucleoside complex stabilization by ribonuclease A demonstrates phosphate mimicry.

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