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J Biol Chem, Vol. 273, Issue 36, 23353-23360, September 4, 1998

Genetic Complexity, Structure, and Characterization of Highly Active Bovine Intestinal Alkaline Phosphatases

Thomas Manes^§, Marc F. Hoylaerts^¶, Rainer Müller, Friedrich Lottspeich^**, Werner Hölke, and José Luis Millán

From the  Department of Medical Genetics, Umeå University, S-901 85 Umeå, Sweden, ^¶ Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium,  Boehringer Mannheim GmbH, D-82377 Penzberg, Germany, ^** Max-Planck Institute for Biochemistry, D-82152 Martinsried, Germany, and ^§ The Burnham Institute, La Jolla, California 92037

Mammalian alkaline phosphatases (APs) display 10-100-fold higher k_cat values than do bacterial APs. To begin uncovering the critical residues 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, ~3,000 units/mg) previously cloned in our laboratory, and a highly active isozyme (bIAP II, ~8,000 units/mg) of hitherto unknown sequence. An unprecedented level of complexity was revealed for the bovine AP family of genes during our attempts to clone the bIAP II cDNA from cow intestinal RNAs. We cloned and characterized two novel full-length IAP cDNAs (bIAP III and bIAP IV) and obtained partial sequences for three other IAP cDNAs (bIAP V, VI, and VII). Moreover, we identified and partially cloned a gene coding for a second tissue nonspecific AP (TNAP-2). However, the cDNA for bIAP II, appeared unclonable. The sequence of the entire bIAP II isozyme was determined instead by a classical protein sequencing strategy using trypsin, carboxypeptidase, and endoproteinase Lys-C, Asp-N, and Glu-C digestions, as well as cyanogen bromide cleavage and NH₂-terminal sequencing. A chimeric bIAP II cDNA was then constructed by ligating wild-type and mutagenized fragments of bIAP I, III, and IV to build a cDNA encoding the identified bIAP II sequence. Expression and enzymatic characterization of the recombinant bIAP I, II, III, and IV isozymes revealed average k_cat values of 1800, 5900, 4200, and 6100 s¹, respectively. Comparison of the bIAP I and bIAP II sequences identified 24 amino acid positions as likely candidates to explain differences in k_cat. Site-directed mutagenesis and kinetic studies revealed that a G322D mutation in bIAP II reduced its k_cat to 1300 s¹, while the converse mutation, i.e. D322G, in bIAP I increased its k_cat to 5800 s¹. Other mutations in bIAP II had no effect on its kinetic properties. Our data clearly indicate that residue 322 is the major determinant of the high catalytic turnover in bovine IAPs. This residue is not directly involved in the mechanism of catalysis but is spatially sufficiently close to the active site to influence substrate positioning and hydrolysis of the phosphoenzyme complex.

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