Reversible inhibition of intestinal alkaline phosphatase by inositol hexaphosphate and its Cu(II) coordinate complexes

Abstract

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 K i values in the μM range. Without added inhibitors present, the kinetic parameters are k cat 5.7 × 10 3 min −1; and K M, 18 μM. In the presence of 62 μM IHP, k cat was essentially unchanged with an apparent K M of 68 μM giving a K i of 22 μM. In the presence of an (IHP-Cu) complex (62 μM IHP, 128 μM Cu(II)), the apparent K M was 55 μM and K i was 30 μM. At a ratio of Cu(II):IHP of 6.0 (372:62 μM) the apparent K M was 30 μM and K i was 94 μM. 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.