Divergent N-Terminal Regions in AMP Deaminase and Isoform-Specific Catalytic Properties of the Enzyme

AMP deaminase (AMPD) catalyzes the hydrolytic deamination of AMP to IMP and NH3. This activity is represented throughout mammalian tissues and cells by at least three isoforms. Human AMPD cDNAs have been cloned and sequenced, leading to predictions that each isoform contains distinct amino ends (N-terminal regions) in contrast to their highly conserved carboxyl-ends (C-terminal regions). Wild type, truncated, and chimeric human ARPD1 (isoform M) and AMPD2 (isoform L) cDNAs were expressed and the resultant activities partially characterized as a means to examine the role of divergent N-terminal regions in these polypeptides (residues 1-262 and 1-258 of isoforms M and L, respectively) on isoform-specific catalytic properties. Similar to activities purified from human tissues, in the presence of monovalent cation, wild type isoform M displayed hyperbolic kinetics in the presence and absence of ATP, whereas wild type isoform L, exhibited allosteric activation in the presence of this nucleotide effector. Expression of both a chimeric M (5′-AMPD1)/L (3′-AMPD2) construct and one in which the N-terminal region of isoform L was deleted produced activities that were also allosterically regulated by ATP. However, no AMPD activity was detectable following expression of either a chimeric L (5′-AMPD2)/M (3′-ARPD1) construct or one in which the N-terminal region of isoform RI had been deleted. The N-terminal region also affected the relative ability of each recombinant AMPD activity to deaminate substrate analogs modified in either the sugar or the phosphate, but not in the purine base, moieties of AMP. These combined data show (i) that isoform M, but not isoform L, absolutely requires its N-terminal region for proper function, (ii) that the C-terminal region of isoform L is responsible for allosteric activation by ATP, (iii) an effect of the N-terminal on substrate-enzyme interaction, a contention that is discussed in context with available information regarding the related purine catabolic activity, adenosine deaminase.