Reversible inhibition of intestinal alkaline phosphatase by inositol hexaphosphate and its Cu(II) coordinate complexes.
The influence of inositol hexakisphosphate (IHP) and its cupric ion chelate complexes on alkaline phosphatase (APase) catalysis of p-nitrophenyl phosphate hydrolysis at pH 7.2 has been determined. Both IHP and (IHP-Cu) complexes, but not Cu(II) alone, are effective inhibitors of the enzyme and are of the strictly competitive type with Ki values in the microM range. Without added inhibitors present, the kinetic parameters are kcat 5.7 x 10(3) min(-1); and KM, 18 microM. In the presence of 62 microM IHP, kcat was essentially unchanged with an apparent KM of 68 microM giving a Ki of 22 microM. In the presence of an (IHP-Cu) complex (62 microM IHP, 128 microM Cu(II], the apparent KM was 55 microM and Ki was 30 microM. At a ratio of Cu(II):IHP of 6.0 (372:62 microM) the apparent KM was 30 microM and Ki was 94 microM. The inhibitory effect of (IHP-Cu) complexes thus decreases as the IHP binding sites for cupric ions become saturated. A high ionic strength environment markedly reduces the inhibitory effect of IHP. Previous studies have also shown that rates of APase inactivation by (IHP-Cu) complexes are also ionic strength sensitive [1]. The inhibition of APase activity by either IHP or its coordinate complexes with cupric ions is evidence for their interaction at the enzyme's catalytic sites. Such results thus provide support for an essential element of the mechanism previously suggested for the reversible inactivation (as opposed to inhibition) of APase by (IHP-Cu) chelate complexes, viz., that it may be due to a metal ion exchange reaction leading to the formation of a Cu(II)-substituted enzyme.
Martin
C J
CJ
Department of Biochemistry, Chicago Medical School, University of Health Sciences, Illinois 60064.
Evans
W J
WJ
D000469:Alkaline Phosphatase / Q000037:antagonists & inhibitors*; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002417:Cattle; D003300:Copper / Q000494:pharmacology*; D007413:Intestinal Mucosa / Q000201:enzymology*; D007700:Kinetics; D010833:Phytic Acid / Q000494:pharmacology*
D003300:Copper; D010833:Phytic Acid; D000469:Alkaline Phosphatase