Regulation of skeletal muscle AMP deaminase: lysine residues are critical for the pH-dependent positive homotropic cooperativity behaviour of the rabbit enzyme

Reaction of rabbit skeletal muscle AMP deaminase with a low molar excess of trinitrobenzene sulfonic acid (TNBS) results ill conversion of the enzyme into a species with about six trinitrophenylated lysine residues per molecu le which no longer manifests positive homotropic cooperativity at pH 7.1 or at the optimal pH value of 6.5 in the presence of low K+ concentrations. S ubstitution of the reactive thiol groups with 5,5'-dithiobis-(2-nitrobenzoi c acid) does not protect the enzyme from the TNBS-induced changes of the ca talytic properties, indicating that cysteine residues modification is not a t the basis of the effects of TNBS treatment on AMP deaminase and strongly suggesting the obligatory participation of lysine residues to the constitut ion of a regulatory anionic site to which AMP must bind to stimulate the en zyme at alkaline pH. The TNBS-troated enzyme is also completely desensitize d to inhibition by ATP, but not to inhibition by GTP and stimulation by ADP . This observation suggests a connection between the operation of the hypot hesized anionic activating site, responsible for positive homotropic cooper ativity, and the inhibition exerted by anionic compounds that compete for t he same site, among them the most efficient metabolite being probably ATP. (C) 2001 Elsevier Science B.V. All rights reserved.