Kinetic studies of the effect of addition of Co2+ or Mn2+ to a highly purified
dipeptidase from Ehrlich-Lettré mouse
ascites tumor cells show that these metals specifically activate the hydrolyses of certain classes of
dipeptides. This
enzyme was previously (S. Hayman and E. K. Patterson, 1971, J. Biol. Chem. 246, 660) reported to be a Zn-metalloenzyme. It is now shown that Zn is the only
metal that can partially restore the activity of the
EDTA-inhibited
dipeptidase in cleaving
Ala-Gly. Addition of Co2+ increases the Vmax of N-terminal Gly-
dipeptides with increase in Km while addition of Mn2+ primarily activates the hydrolysis of
Pro-Gly, again with increases in both Vmax and Km. Prior incubation (5 min, 30 degrees) of the
dipeptidase with the appropriate
metal ions causes decrease in initial lag time in the Co2+-activated hydrolysis of Gly-Gly and the Mn2+-activated hydrolysis of
Pro-Gly. Long-term (6-19 hr, 0 degrees) incubation of the
enzyme with Co2+ results in loss of activity toward
Ala-Gly with a concomitant 13-fold increase in the rate of Gly-Gly hydrolysis and loss of 70% of the Zn2+ from the
dipeptidase; these effects can be partially reversed by addition of Zn2+. In contrast, long-term incubation of the
enzyme with Mn2+ results in no loss of Zn2+ and a twofold increase in activity toward
Pro-Gly. One affinity constant of 1.4 muM for Co2+ and two constants of 0.23 and 27 muM for Mn2+ were determined by kinetic experiments. Comparison of the properties of this
tumor enzyme with a
dipeptidase purified in our laboratory from Escherichia coli B, and with mammalian
dipeptidases highly purified by others, shows remarkable similarities in molecular weights and molecular activities toward the preferred substrates but in the case of bacterial
dipeptidase, differences in substrate specificities and in the effect of
metal ions.