kinetics and binding studies
Springer Online Journal Archives 1860-2000
Chemistry and Pharmacology
Abstract Kinetics and binding studies of RNase A and its natural polymeric substrate (RNA), as well as the natural mixture of free 3′-ribonucleotides, were performed by difference spectrophotometry. The obtained kinetic saturation curve, with an anomalous nonhyperbolic shape and a distinct transition point, showed the interchange between the two conformational forms of the enzyme. This occurred in a narrow range of substrate concentration. At low substrate concentration, in spite of the existence of one catalytic cleft, RNase A behaves as a cooperative system, perhaps due to the interactions among the four cooperative binding subsites in the active cleft. At high substrate concentration, the conformational change did occur and was accompanied by a decrease in cooperativity and increment of the catalytic constant. The multiphasic shape of the binding curve, which, in the presence of the enzyme, produced 3′-ribonucleotides (as the ligand molecules), shows four binding subsites. The first three subsites are specific for the attachment of phosphate, ribose, and base moieties belonging to the first bound 3′-ribonucleotide in the direction of 3′-phosphate → ribose → base-5′. The fourth subsite relates to the second phosphate group of the second bound 3′-ribonucleotide. The binding direction also converts to 5′-phosphate → ribose → base-3′ for the ribonucleotide monomers in the RNA structure.
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