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 concentrations. We have investigated these kinetics by determining 3'-CMP formation both spectrophotometrically and by a highly specific and quantitative chemical sampling method. With the use of RNase A derivatives that lack a functional p2 binding subsite, evidence is presented that the abnormal kinetics with the native enzyme are caused by the sequential binding of the substrate to the several subsites that make up the 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 p2 binding subsite are used. 2) The kcat/Km values with oligocytidylic acids of increasing lengths (ending in C>p) show a behavior that parallels the specific velocity with C>p at high concentrations: increase 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 increases the kcat obtained with a fixed concentration of C>p as substrate. 4) High concentrations of C>p protect the enzyme from digestion with subtilisin, which results in a more compact molecule, implying large substrate concentration-induced conformational changes. The data for the hydrolysis of C>p by RNase A can be fitted to a fifth order polynomial that has been derived from a kinetic scheme based on the sequential binding of several monomeric substrate molecules.
Moussaoui
M
M
Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain.
Nogués
M V
MV
Guasch
A
A
Barman
T
T
Travers
F
F
Cuchillo
C M
CM
D000241:Adenosine / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002417:Cattle; D003568:Cytidine Monophosphate / Q000378:metabolism; D003597:Cytosine Nucleotides / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D015394:Molecular Structure; D009154:Mutation / Q000235:genetics; D009843:Oligoribonucleotides / Q000378:metabolism; D011066:Poly C / Q000378:metabolism; D011487:Protein Conformation; D012259:Ribonuclease, Pancreatic / Q000737:chemistry*; D013053:Spectrophotometry; D013379:Substrate Specificity; D013381:Subtilisins / Q000378:metabolism
D003597:Cytosine Nucleotides; D009843:Oligoribonucleotides; D011066:Poly C; C019310:cifostodine; D012259:Ribonuclease, Pancreatic; D013381:Subtilisins; D003568:Cytidine Monophosphate; D000241:Adenosine