A comparative analysis of kinetic data for fungal beta-galactosidases has revealed that the ratio of Michaelis constants (MCR) with two substrates (lactose and o/p-nitrophenyl-beta-D-galactopyranoside. NPG) is approximately constant for certain groups of enzymes. It equals 35 +/- 3 and 10 +/- 1.5 for the mould and the yeast enzymes, respectively. MCR = K-M(lactose)/K-M(NPG) Thus, MCR provides a simple and fast method for identification of related enzymes. MCR for a group of enzymes with a pair of standard substrates may serve as an identification number of this group. A substantial deviation of MCR for a particular enzyme from the group parameter might be an indication of possible errors in K-M determination, or in assignment of the enzyme to this group. An important advantage of this approach is that the experimental K-M values may be obtained for enzymes with different purity and concentration. Within the framework of classical Michaelis-Menten kinetics and in accordance with the customary use of K-M for a comparison of enzyme-substrate affinity, the MCR parameter may be interpreted as a relative substrate affinity. This proportion remains nearly the same for different mould (or yeast) beta-galactosidases. though the affinity to a particular substrate may vary essentially from enzyme to enzyme. The constancy of MCR may be a manifestation of structural similarity of binding sites for these enzymes. (C) 2004 Elsevier Inc. All fights reserved.