Isothermal Titration Calorimetric Study of RNase-A Kinetics (cCMP 3'-CMP) Involving End-Product Inhibition

Authors: Shawn D. Spencer¹; Robert B. Raffa²

Source: Pharmaceutical Research, Volume 21, Number 9, September 2004 , pp. 1642-1647(6)

Publisher: Springer

Abstract:

Purpose. Isothermal titration calorimetry (ITC) and progress curve analysis was used to measure the enzyme kinetic parameters (K_M and K_cat) 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 l cCMP (20 mM) into bovine pancreatic RNase-A (600 nM) in 50 mM Na⁺ acetate buffer (pH 5.5; 37°C) was monitored for 1500-2000 s. Thermal power (dQ/dt), equal to (1) /H_app × d(cCMP)/dt was recorded every 1 s. The end-product inhibition constant (K_p) 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 K_p to yield K_M and K_cat.

Results. H_app for each run was obtained by integration of the progress curve. The plot of -d[cCMP]/dt against [cCMP] yielded the kinetic parameters K_M = 105.3 M, 121.6 M, and 131.3 M; K_cat = 1.63 s^-1, 1.56 s^-1, and 1.71 s^-1. The end-product bound with 1:1 stoichiometry and K_p = 53.2 M.

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.

Keywords: calorimetry; end-product inhibition; enzyme kinetics; RNase

Document Type: Research article

DOI: http://dx.doi.org/10.1023/B:PHAM.0000041460.78128.0f

Affiliations: 1: Department of Pharmaceutical Sciences, School of Pharmacy, Temple University, Philadeplhia, Pennsylvania 19140, USA 2: Department of Pharmaceutical Sciences, School of Pharmacy, Temple University, Philadeplhia, Pennsylvania 19140, USA; Department of Pharmacology, School of Medicine, Temple University, Philadelphia, Pennsylvania 19140, USA. ( ), Email: robert.raffa@temple.edu

Publication date: 2004-09-01

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