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.
Department of Chemistry, University of British Columbia, Vancouver, Canada.
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Withers
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Rupitz
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