Magnesium in the active site of Escherichia coli alkaline phosphatase is important for both structural stabilization and catalysis.

Site-specific mutagenesis was used to explore the roles of the side chains of residues Lys-328 and Asp-153 in Escherichia coli alkaline phosphatase. The D153H enzyme exhibits a 3.5-fold decrease in activity at pH 8.0 compared to that of the wild-type enzyme, while a double mutant D153H/K328H exhibits a 16-fold decrease in activity under these conditions. However, the Km values for both enzymes, employing the substrate p-nitrophenyl phosphate, are lower than the value for the wild-type enzyme. The Ki for phosphate, which is pH- and Mg(2+)-dependent, is decreased for the D153H enzyme and increased for the D153H/K328H enzyme. Relative to the wild-type enzyme, both mutant enzymes bind Mg2+ more weakly and undergo a time-dependent activation induced by Mg2+. The half-time of the activation process is independent of the Mg2+ concentration, indicating that the activation most probably involves a conformational change. The pH versus activity profiles of both enzymes are altered relative to that of the wild-type enzyme and exhibit greatly enhanced activity, relative to that of the wild-type enzyme, at high pH values. The pre-steady-state kinetics for the D153H and D153H/K328H enzymes exhibit a transient burst of product formation at pH 8.0, under conditions at which the wild-type enzyme exhibits no transient burst, indicating that at pH 8.0 the hydrolysis of the covalent enzyme-phosphate complex is rate-determining and not the release of phosphate from the noncovalent enzyme-phosphate complex as is observed for the wild-type enzyme. Therefore, these mutations are directly influencing catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Factor V Quebec revisited.

Factor V Quebec has been described as a bleeding disorder that exhibits an autosomal dominant inheritance pattern and presents severe bleeding after trauma. Two members of a fourth-generation (IV.13 and IV.15) Canadian family have been studied in detail and are the subject of this report. Their clinical presentations and histories have been described previously (Tracy et al: J Clin Invest 74:1221, 1984). Persistent abnormalities include mild thrombocytopenia and defective platelet factor V. Plasma factor V is present at near normal concentration and is fully functional. Thus, the bleeding diathesis appears to reflect the absence of platelet factor V activity. The recent report (Hayward et al: Blood 84:110a, 1994 [suppl, abstr]) of multimerin deficiency in these individuals led us to reevaluate these patients. Western blot analyses of platelet lysates developed with a variety of monoclonal antibodies show that the alpha-granule proteins, fibrinogen, von Willebrand factor, factor V and osteonectin are decreased in concentration and significantly degraded in the platelets of these patients. Thrombospondin, while not degraded, is substantially decreased. In contrast, platelet factor 4 and beta-thromboglobulin do not appear to be affected. These observations suggest that the alpha-granules are correctly assembled but the contents are subsequently subjected to proteolytic degradation. The results indicate that factor V Quebec disorder is probably associated with a generalized defect that leads to degradation of most proteins of the alpha-granules.