The number of functional active sites per molecule of the adenosylcobalamin-dependent enzyme, ethanolamine ammonia-lyase, as determined by a kinetic method.
1. A kinetic approach to the determination of the number of functional active sites per molecule of the adenosylcobalamin-dependent enzyme, ethanolamine ammonia-lyase, is described. 2. Time courses for formation and breakdown of a cob(II)alamin intermediate during reaction of the enzyme, fully saturated with adenosylcobalamin, with L-2-aminopropanol as substrate, were followed using a stopped-flow spectrophotometer under two conditions: (a) enzyme concentration much greater than that of substrate, (b) substrate concentration much greater than that of enzyme. 3. Results were analysed in terms of a three-step mechanism involving binding of substrate (k+1 step), cob(II)alamin formation (k+2 step) and cob(II)alamin breakdown (k+3 step). the kinetic scheme was shown to be sufficient to account for the observed time courses and rate constants of 80 s-1 (k+2) and 1.5 s-1 (k+3) were determined. 4. The number of active sites per enzyme molecule (n) was calculated from the kinetic data in three ways: (a) calculation from amplitude of absorbance measurement, (b) calculation from measurements of the values of rate constants and (c) analysis by computation of the kinetic data using the computer program FACSIMILE. A value for n close to 6 was calculated by each of these methods. This value is in disagreement with the literature value of about two sites per molecule but is consistent with the I6II6 subunit structure of the enzyme. 5. Kinetic analysis of data from experiments in which the adenosylcobalamin concentration was varied while substrate and enzyme concentrations remained constant showed that all the active sites function with identical rate constants. 6. The principle and mathematical basis of the kinetic method for determining the value of n is given as an Appendix.
D000642:Ammonia-Lyases / Q000378:metabolism*; D001665:Binding Sites; D003013:Clostridium / Q000201:enzymology; D003038:Cobamides / Q000494:pharmacology*; D003201:Computers; D004981:Ethanolamine Ammonia-Lyase / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D011412:Propanolamines / Q000494:pharmacology
D003038:Cobamides; D011412:Propanolamines; D000642:Ammonia-Lyases; D004981:Ethanolamine Ammonia-Lyase; C000913:cobamamide