Measurement of active-site homology between potato and rabbit muscle alpha-glucan phosphorylases through use of a linear free energy relationship.
The Michaelis-Menten parameters (Vmax and Km) for turnover of an extensive series of deoxy and deoxyfluoro derivatives of alpha-D-glucopyranosyl phosphate by the alpha-glucan phosphorylase from potato tuber have been determined. Very large rate reductions are observed as a consequence of each substitution, primarily due to losses in specific binding interactions, most likely hydrogen bonding, at the enzymic transition state. Comparison of the Vmax/Km values so determined with those measured for rabbit muscle alpha-glucan phosphorylase [Street et al. (1989) Biochemistry 28, 1581] reveals an astonishingly similar specificity, especially in light of the phylogenetic separation of their host organisms. This indicates that very similar hydrogen-bonding interactions between the enzyme and the substrate must be present at the transition states for the two enzymic reactions; therefore, they have very similar active sites. Quantitation of this similarity is achieved by plotting the logarithm of the Vmax/Km value for each substrate analogue with the potato enzyme against the same parameter for the muscle enzyme, yielding straight lines (p = 0.998 and 0.999) of slope 1.0 and 1.2 for the deoxy and deoxyfluoro substrates, respectively. Since the correlation coefficient of such plots is a direct measure of the similarity of the two transition-state complexes, thus of the enzyme active sites, it can be used as a measure of active-site homology between the two enzymes. The extremely high homology observed in this case is consistent with the observed sequence homology at the active site.
Withers
S G
SG
Department of Chemistry, University of British Columbia, Vancouver, Canada.
Rupitz
K
K
D000818:Animals; D001665:Binding Sites; D005075:Biological Evolution; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D005958:Glucosephosphates / Q000378:metabolism; D006860:Hydrogen Bonding; D007700:Kinetics; D009132:Muscles / Q000201:enzymology*; D006005:Phosphorylases / Q000235:genetics* / Q000378:metabolism; D011817:Rabbits / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D011198:Solanum tuberosum / Q000235:genetics*; D013045:Species Specificity
D005958:Glucosephosphates; D006005:Phosphorylases