STRUCTURE-ACTIVITY RELATIONSHIP OF CYTOPLASMIC 5-NUCLEOTIDASE SUBSTRATE SITES

Various 5'-nucleotidases (EC 3.1.3.5) exist in vertebrate tissues. The sequence and cDNA. cloning of the membrane-bound ecto-5'-nucleotidase (e-N) and one of the cytosolic isoenzymes, IMP-preferring (c-N-II), b ut not the cytosolic AMP-preferring form (c-N-I), have been reported. While c-N-II has a broad tissue distribution, c-N-I is found only in v ertebrate heart, The published data on substrate specificity involve m ainly the naturally occurring nucleoside monophosphates, without a sys tematic structure-activity relationship study. In the present study we have used a series of AMP and IMP analogues to examine the structure- activity relationship for c-N-I and c-N-II in detail. The rank order o f activity of the test compounds differed substantially between c-N-I and c-N-II. c-N-I and c-N-II varied with respect to the following inte ractions with substrate: (1) hydrogen-bond formation with the substitu ent in the 6-position of the purine ring (a donor-type with c-N-I and an acceptor-type with c-N-II); and (2) hydrophobic attraction of the 6 -position unsubstituted purine ring (more pronounced with c-N-I than w ith c-N-II). No better substrate than 5'-AMP was found for c-N-I. We p ropose that c-N-I functions as an AMP-binding protein in the myocardia l cell with an important role during ischaemic ATP breakdown when AMP accumulates rapidly.