Acetohydroxamate inhibition of the activity of urease from dehusked seeds of water melon (Citrullus vulgaris)

Abstract

Urease from the seeds of watermelon (Citrullus vulgaris) was purified to apparent homogeneity, using two acetone fractionation steps, heat treatment at 48°C and gel filtration through Sephadex G-200. Effect of aceto-hydroxamic acid (AHA) on the activity of the homogeneous enzyme preparation (sp. act. 3000 ± 550 U/mg protein) was investigated. AHA exhibited a concentration-dependent inhibition both in the presence and absence of the substrate. The inhibition was uncompetitive and the Ki was 2.5 mM. Binding of AHA with the enzyme was reversible, as 63% activity could be restored by dialysis. Time-dependent inhibition revealed a monophasic inhibition of the activity. Addition of β-mercaptoethanol (ME) gradually abolished the inhibition. Pre-treatment of native enzyme with 8.0 mM ME for 5 min at 30°C exhibited protection against AHA-induced inhibition. The significance of these observations is discussed. © 2004 Taylor & Francis Ltd.