GENETIC COMPLEXITY, STRUCTURE, AND CHARACTERIZATION OF HIGHLY-ACTIVE BOVINE INTESTINAL ALKALINE PHOSPHATASES

Mammalian alkaline phosphatases (APs) display 10-100-fold higher k(cat ) values than do bacterial APs. To begin uncovering the critical resid ues that determine the catalytic efficiency of mammalian APs, we have compared the sequence of two bovine intestinal APs, i.e. a moderately active isozyme (bovine intestinal alkaline phosphatase, bIAP I, simila r to 3,000 units/mg) previously cloned in our laboratory, and a highly active isozyme (bIAP II, similar to 8,000 units/mg) of hitherto unkno wn sequence. An unprecedented level of complexity was revealed for the bovine AP family of genes during our attempts to clone the bIAP II cD NA from cow intestinal RNAs. We cloned and characterized two novel ful l-length IAP cDNAs (bIAP III and bIAP IV) and obtained partial sequenc es for three other IAP cDNAs (bIAP V, VI, and VII). Moreover, we ident ified and partially cloned a gene coding for a second tissue nonspecif ic AP (TNAP-2). However, the cDNA for bIAP II, appeared unclonable. Th e sequence of the entire bIAP II isozyme was determined instead by a c lassical protein sequencing strategy using trypsin, carboxypeptidase, and endoproteinase Lys-C, Asp-N, and Glu-C digestions, as well as cyan ogen bromide cleavage and NH2-terminal sequencing. A chimeric bIAP II cDNA was then constructed by ligating wildtype and mutagenized fragmen ts of bIAP I, III, and IV to build a cDNA encoding the identified bIAP II sequence. Expression and enzymatic characterization of the recombi nant bIAP I, II, III, and TV isozymes revealed average k(cat) values o f 1800, 5900, 4200, and 6100 s(-1), respectively. Comparison of the bI AP I and bIAP II sequences identified 24 amino acid positions as likel y candidates to explain differences in k(cat). Site-directed mutagenes is and kinetic studies revealed that a G322D mutation in bIAP II reduc ed its k(cat) to 1300 s(-1), while the converse mutation, ie. D322G, i n bIAP I increased its k(cat) to 5800 s(-1). Other mutations in bIAP I I had no effect on its kinetic properties. Our data clearly indicate t hat residue 322 is the major determinant of the high catalytic turnove r in bovine IAPs. This residue is not directly involved in the mechani sm of catalysis but is spatially sufficiently close to the active site to influence substrate positioning and hydrolysis of the phosphoenzym e complex.