MOLECULAR-CLONING OF DIADENOSINE TETRAPHOSPHATASE FROM PIG SMALL-INTESTINAL MUCOSA AND IDENTIFICATION OF SEQUENCE BLOCKS COMMON TO DIADENOSINE POLYPHOSPHATE HYDROLASES AND PHOSPHORYLASES

Diadenosine 5',5 '''-P-1,P-4-tetraphosphate (Ap(4)A) pyrophosphohydrol ase is the enzyme responsible for reducing intracellular levels of the stress-responsive nucleotide diadenosine 5',5 '''-P-1,P-4-tetraphosph ate. In order to gain more information on the relationships between th e enzymes hydrolysing diadenosine polyphosphates in different eukaryot es, the Ap(4)A hydrolase and a corresponding cDNA have been isolated f rom pig small intestinal mucosa by standard procedures. The enzyme is a typical mammalian Ap(4)A hydrolase (K-m = 0.8 mu M), being sensitive to inhibition by fluoride (K-i = 24 mu M) and adenosine 5'-tetraphosp hate (K-i = 10 nM) and yielding ATP and AMP as products. A low K-m Ap( 4)A hydrolase (K-m = 0.3 mu M) was also isolated from rabbit small int estinal mucosa. These enzymes differ from the rat intestinal mucosal h ydrolase, which has much higher values of K-m for Ap(4)A and K-i for a denosine 5'-tetraphosphate. A cDNA encoding the pig enzyme was isolate d from a pig ileum cDNA library. The derived amino acid sequence of th e 16.8 kDa gene product shows 88% identity and 96% similarity to that of the human enzyme. The sequence has the same modification of the Mut T motif found in the human enzyme in which a threonine residue replace s a hydrophobic amino acid. Sequence comparisons among eukaryotic diad enosine polyphosphate hydrolases and phosphorylases reveal two blocks of amino acid similarity, including a motif, Z[AD]Gx[ED]JAGQ, which ma y be involved in polyphosphate binding by the hydrolases, and an invar iant histidine residue that may be involved in catalysis. These sequen ce similarities may have arisen by convergent evolution. (C) 1997 Else vier Science Ltd.