Interaction of TaqI endonuclease with the phosphate backbone. Effects of stereospecific phosphate modification.
The restriction endonuclease TaqI exhibits extreme specificity for its cognate sequence, TCGA, but a direct hydrogen bond readout model fails to account for this property. The present study examines the role of phosphate contacts in the enzyme-substrate and transition state complexes. An S-methyl group was introduced into each of the pTpCpGpApNpN internucleotide linkages using a hybrid chemical-enzymatic synthesis, in which sulfur substitutions of nonbridging phosphate oxygens directed the placement of methyl groups. The resulting 12 diastereomerically pure phosphate-modified substrates were tested for binding and cleavage by TaqI. The largest binding effects were induced by pro-Sp methylations at the pTpCpGA phosphates, which destabilized the enzyme-substrate complex by 1.0-1.6 kcal/mol. Cleavage of the modified strand was inhibited completely by modifications at the TpCpGpA phosphates and inhibited significantly at the TCGApNp phosphates. Cleavage of both strands was completely inhibited by modification of the TCGpA linkage. Effects on the cleavage of the unmodified strand were used to implicate phosphate modifications that caused global perturbations in the structure of the transition state complex. These results lend support for a model for the specificity of TaqI, in which sequence-specific phosphate contacts are formed in the transition state, thus amplifying the apparent contribution of base contacts to transition state stabilization.
Department of Microbiology, Cornell University Medical College, New York, New York 10021.
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Mayer
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Barany
D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010755:Organophosphates / Q000737:chemistry / Q000378:metabolism*; D009994:Osmolar Concentration; D010100:Oxygen / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity; D013816:Thermodynamics
D010755:Organophosphates; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; C027046:TCGA-specific type II deoxyribonucleases; D010100:Oxygen