Isothermal titration calorimetric study of RNase-A kinetics (cCMP --> 3'-CMP) involving end-product inhibition.
OBJECTIVE
Isothermal titration calorimetry (ITC) and progress curve analysis was used to measure the enzyme kinetic parameters (KM and kcat) of the hydrolysis of cCMP by RNase-A, a reaction that includes end-product competitive inhibition by 3'-CMP.


METHODS
The heat generated from injection of 9-15 microl cCMP (20 mM) into bovine pancreatic RNase-A (600 nM) in 50 mM Na+ acetate buffer (pH 5.5; 37 degrees C) was monitored for 1500-2000 s. Thermal power (dQ/dt), equal to (1)/deltaH(app) x d(cCMP)/dt was recorded every 1 s. The end-product inhibition constant (Kp) and enthalpy of the inhibitor binding interaction was obtained from the saturation data of 60 sequential injections of 3'-CMP (1.2 mM) into 0.05 mM RNase-A. The data of the plot of -d[cCMP]/dt against [cCMP] were fitted to kinetic equations incorporating Kp to yield KM and kcat.


RESULTS
DeltaH(app) for each run was obtained by integration of the progress curve. The plot of -d[cCMP]/dt against [cCMP] yielded the kinetic parameters KM = 105.3 microM, 121.6 microM, and 131.3 microM; kcat = 1.63 s(-1), 1.56 s(-1), and 1.71 s(-1). The end-product bound with 1:1 stoichiometry and Kp = 53.2 microM.


CONCLUSIONS
The combination of progress curve analysis and ITC allowed rapid and facile measurement of the kinetic parameters for catalytic conversion of cCMP to 3'-CMP by RNase-A, a reaction complicated by end-product inhibition.
Spencer
Shawn D
SD
Department of Pharmaceutical Sciences, School of Pharmacy, Temple University, Philadelphia, Pennsylvania 19140, USA.
Raffa
Robert B
RB
D000818:Animals; D002151:Calorimetry / Q000379:methods; D002417:Cattle; D003568:Cytidine Monophosphate / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D003597:Cytosine Nucleotides / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008956:Models, Chemical; D015394:Molecular Structure; D012259:Ribonuclease, Pancreatic / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013816:Thermodynamics
D003597:Cytosine Nucleotides; C019310:cifostodine; D012259:Ribonuclease, Pancreatic; D003568:Cytidine Monophosphate