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 AMPD1 (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'-AMPD1) construct or one in which the N-terminal region of isoform M 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 region on substrate-enzyme interaction, a contention that is discussed in context with available information regarding the related purine catabolic activity, adenosine deaminase.
Bausch-Jurken
M T
MT
Department of Cell Biology and Anatomy, Medical College of Wisconsin, Milwaukee 53226, USA.
Sabina
R L
RL
D000659:AMP Deaminase / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D006801:Humans; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism*; D015183:Restriction Mapping; D017384:Sequence Deletion
D007527:Isoenzymes; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D000659:AMP Deaminase