THE SUBSITES STRUCTURE OF BOVINE PANCREATIC RIBONUCLEASE-A ACCOUNTS FOR THE ABNORMAL KINETIC-BEHAVIOR WITH CYTIDINE 2',3'-CYCLIC PHOSPHATE

The kinetics of the hydrolysis of cytidine 2',3'-cyclic phosphate (C>p ) to 3'-CMP by ribonuclease A are multiphasic at high substrate concen trations. We have investigated these kinetics by determining 3'-CMP fo rmation both spectrophotometrically and by a highly specific and quant itative chemical sampling method. With the use of RNase A derivatives that lack a functional p(2) binding subsite, evidence is presented tha t the abnormal kinetics with the native enzyme are caused by the seque ntial binding of the substrate to the several subsites that make up th e active site of ribonuclease, The evidence is based on the following points. 1) Some of the unusual features found with native RNase A and C>p as substrate disappear when the derivatives lacking a functional p (2) binding subsite are used. 2) The k(cat)/K-m values with oligocytid ylic acids of increasing lengths tending in C>p) show a behavior that parallels the specific velocity with C>p at high concentrations: incre ase in going from the monomer to the trimer, a decrease from tetramer to hexamer, and then an increase in going to poly(C), 3) Adenosine inc reases the k(cat) obtained with a fixed concentration of C>p as substr ate. 4) High concentrations of C>p protect the enzyme from digestion w ith subtilisin, which results in a more compact molecule, implying lar ge substrate concentration-induced conformational changes. The data fo r the hydrolysis of C>p by RNase A can be fitted to a fifth order poly nomial that has been derived from a kinetic scheme based on the sequen tial binding of several monomeric substrate molecules.