Effects of allosteric activation on the primary and secondary kinetic isotope effects for three AMP nucleosidases.
Kinetic isotope effects (V/K) were measured with AMP nucleosidases isolated from Azotobacter vinelandii, from a Vmax mutant enzyme of A. vinelandii and from Escherichia coli. Specifically labeled AMP substrates were used to measure 3H secondary and 14C primary kinetic isotope effects on the N-glycosidic bond hydrolysis of AMP in the presence and absence of the allosteric activator, MgATP. Use of the three enzymes, variable MgATP concentration, a poor substrate (dAMP), and variable pH has allowed determination of the isotope effects over a 5000-fold range in the catalytic turnover number. The primary kinetic isotope effects were 1.025 +/- 0.004 and 1.041 +/- 0.006 for the native A. vinelandii enzyme and mutant enzyme, respectively, and were independent of MgATP concentration. The E. coli AMP nucleosidase had a primary isotope effect of 1.019 +/- 0.003 which was also independent of MgATP concentration. The secondary kinetic isotope effect decreased from 1.066 +/- 0.003 to 1.045 +/- 0.002 for the native enzyme from A. vinelandii as the concentration of MgATP increased from 0 to 500 microM. The secondary isotope effect of the mutant enzyme remained constant at 1.088 +/- 0.005 as the MgATP concentration increased from 0 to 500 microM. The secondary isotope effect of the E. coli enzyme showed a similar pattern to that of the native enzyme, decreasing from 1.087 +/- 0.003 to 1.050 +/- 0.003 as the enzyme was saturated with MgATP at a constant concentration of AMP. Saturation with AMP in the absence of MgATP gave similar results and suggested that AMP can cause the allosteric transition. Both the primary and secondary isotope effects for the native enzyme from A. vinelandii remained constant as the pH was varied in the absence of MgATP. Secondary isotope effects with a poor substrate, dAMP, were 1.08 for both the mutant and wild type enzymes from A. vinelandii in the presence of allosteric activator. In the native enzyme, this isotope effect was independent of MgATP concentration. The relative insensitivity in the magnitude of observed isotope effects to pH, allosteric activator, the mutant enzyme, and a poor substrate (dAMP) indicate that intrinsic isotope effects are being expressed. The data are interpreted in terms of a single rate-limiting transition state for hydrolysis of the N-glycosidic bond, although other mechanisms cannot be eliminated. Using this model, the transition states of the native A. vinelandii and E. coli enzymes exhibit properties of both dissociative and associative mechanisms but become more associative as the allosteric activator becomes saturating.(ABSTRACT TRUNCATED AT 400 WORDS)
D000255:Adenosine Triphosphate / Q000378:metabolism; D001395:Azotobacter / Q000201:enzymology; D002247:Carbon Isotopes; D003839:Deoxyadenosines / Q000378:metabolism; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007553:Isotope Labeling; D007700:Kinetics; D008970:Molecular Weight; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D014316:Tritium
D002247:Carbon Isotopes; D003839:Deoxyadenosines; D014316:Tritium; D000255:Adenosine Triphosphate; D009699:N-Glycosyl Hydrolases; C019699:AMP nucleosidase