A thermodynamic cycle is used to describe barnase catalysis, which considers explicitly the presence of different ionic states of the catalytic residues Glu-73 and His-102 in barnase during the enzyme-substrate recognition process. Reinterpretation of published experimental data using rate equations derived from this cycle provides estimates of the ionization constants of these catalytic side chains, in the free enzyme and in the barnase-GpA complex. In addition, the electrostatic properties of the barnase-d(CGAC) crystal complex and of a barnase-5'3'(AAGAAp)-O-methyl ester modeled complex are investigated by means of a continuum approach to account for solvent polarization effects. Taking GpA as a reference substrate, it is shown that increasing the length of the bound nucleotide induces pK(a) shifts in the catalytic side chains, which modulate the fraction of enzyme in the correct ionic form for achieving the transesterification reaction. The computed results are in good agreement with the experimental variation of the optimum pH of barnase activity. The present analysis underscores the influence of pH effects on the k(cat) and K(M) kinetic constants of barnase and provides the basic formalism for linking the effective kinetic parameters, which usually depend on the pH, to the theoretical estimates of the true kinetic constants.
|Journal||Proteins: Structure, Function and Genetics|
|Publication status||Published - 26 Jul 1996|
- enzyme catalysis
- microbial RNases
- pK(a) shift
- protein-nucleic acid interaction