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A two-step nucleotide-flipping mechanism enables kinetic discrimination of DNA lesions by AGT.

ABSTRACT: O(6)-alkylguanine-DNA alkyltransferase (AGT) repairs damage to the human genome by flipping guanine and thymine bases into its active site for irreversible transfer of alkyl lesions to Cys-145, but how the protein identifies its targets has remained unknown. Understanding molecular recognition in this system, which can serve as a paradigm for the many nucleotide-flipping proteins that regulate genes and repair DNA in all kingdoms of life, is particularly important given that inhibitors are in clinical trials as anticancer therapeutics. Computational approaches introduced recently for harvesting and statistically characterizing trajectories of molecularly rare events now enable us to elucidate a pathway for nucleotide flipping by AGT and the forces that promote it. In contrast to previously proposed flipping mechanisms, we observe a two-step process that promotes a kinetic, rather than a thermodynamic, gate-keeping strategy for lesion discrimination. Connection is made to recent single-molecule studies of DNA-repair proteins sliding on DNA to understand how they sense subtle chemical differences between bases efficiently.

SUBMITTER: Hu J

PROVIDER: S-EPMC2290773 | biostudies-literature | 2008 Mar

REPOSITORIES: biostudies-literature

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A two-step nucleotide-flipping mechanism enables kinetic discrimination of DNA lesions by AGT.

Hu Jie J Ma Ao A Dinner Aaron R AR

Proceedings of the National Academy of Sciences of the United States of America 20080319 12

O(6)-alkylguanine-DNA alkyltransferase (AGT) repairs damage to the human genome by flipping guanine and thymine bases into its active site for irreversible transfer of alkyl lesions to Cys-145, but how the protein identifies its targets has remained unknown. Understanding molecular recognition in this system, which can serve as a paradigm for the many nucleotide-flipping proteins that regulate genes and repair DNA in all kingdoms of life, is particularly important given that inhibitors are in cl ...[more]

PMID: 18353991

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Project description:Human histidine triad nucleotide binding protein 1 (hHint1) is a member of a ubiquitous and ancient branch of the histidine triad protein superfamily. hHint1 is a homodimeric protein that catalyzes the hydrolysis of model substrates, phosphoramidate and acyl adenylate, with a high efficiency. Recently, catalytically inactive hHint1 has been identified as the cause of inherited peripheral neuropathy [Zimon, M., et al. (2012) Nat. Genet. 44, 1080-1083]. We have conducted the first detailed kinetic mechanistic studies of hHint1 and have found that the reaction mechanism is consistent with a double-displacement mechanism, in which the active site nucleophile His112 is first adenylylated by the substrate, followed by hydrolysis of the AMP-enzyme intermediate. A transient burst phase followed by a linear phase from the stopped-flow fluorescence assay indicated that enzyme adenylylation was faster than the subsequent intermediate hydrolysis and product release. Solvent viscosity experiments suggested that both chemical transformation and diffusion-sensitive events (product release or protein conformational change) limit the overall turnover. The catalytic trapping experiments and data simulation indicated that the true koff rate of the final product AMP is unlikely to control the overall kcat. Therefore, a protein conformational change associated with product release is likely rate-limiting. In addition, the rate of Hint1 adenylylation was found to be dependent on two residues with pKa values of 6.5 and 8, with the former pKa agreeing well with the nuclear magnetic resonance titration results for the pKa of the active site nucleophile His112. In comparison to the uncatalyzed rates, hHint1 was shown to enhance acyl-AMP and AMP phosphoramidate hydrolysis by 10(6)-10(8)-fold. Taken together, our analysis indicates that hHint1 catalyzes the hydrolysis of phosphoramidate and acyl adenylate with high efficiency, through a mechanism that relies on rapid adenylylation of the active residue, His112, while being partially rate-limited by intermediate hydrolysis and product release associated with a conformational change. Given the high degree of sequence homology of Hint proteins across all kingdoms of life, it is likely that their kinetic and catalytic mechanisms will be similar to those elucidated for hHint1.

Dataset Information

A two-step nucleotide-flipping mechanism enables kinetic discrimination of DNA lesions by AGT.

Publications

A two-step nucleotide-flipping mechanism enables kinetic discrimination of DNA lesions by AGT.

Similar Datasets

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