IMPORTANCE OF THE REGION AROUND LYSINE-196 FOR CATALYTIC ACTIVITY OF ADENYLYL-CYCLASE FROM ESCHERICHIA-COLI

Escherichia coli adenylyl cyclase contains no sequence that correspond s to the previously defined ATP/GTP binding consensus (A,G)XXXXGK(S,T) . Using a search for lysine residues located adjacent to glycine resid ues, three regions that were possible candidates for part of the ATP b inding site were identified. These were the residues located at positi ons 59, 90, and 196. A plasmid vector capable of overexpressing the cy a gene under the control of the lambda P-L promoter was mutated at the se three loci to convert those lysine residues to methionine. Assays f or catalytic activity of the mutated hyper-expressed proteins revealed that only the mutation at position 196 led to loss of activity. Photo affinity labeling experiments using 8-azido-ATP provided evidence that the loss of activity was associated with a loss of the capability of the enzyme to bind ATP. A further series of replacement mutations in t he hyperexpression vector was created at position 196. Assays of the a denylyl cyclase activity of the mutated proteins showed that replaceme nt of lysine 196 by arginine led to minimal change in the activity. Re placements by histidine, glutamine, or glutamic acid resulted in appro ximately 10-20-fold reductions in the activity; replacements by methio nine, isoleucine, or aspartic acid resulted in total loss of activity. When the mutated forms of the cya gene were expressed under the contr ol of the cya promoter, the activity of the wild-type protein was high er than that of all the mutants, including the arginine replacement mu tant. All of the mutants that retained activity also retained the capa bility of adenylyl cyclase to be stimulated by either inorganic orthop hosphate or GTP. A helical wheel analysis of the region of adenylyl cy clase around lysine 196 revealed a structure compatible with an amphip athic helix with one face enriched with basic amino acid residues. Ass ays for adenylyl cyclase activity of a series of replacement mutations of residues on the hydrophilic face of the helix (R188I, R192I, G195I ) as well as on the hydrophobic face (R197I) indicated that the R188I, G195I, and K196I replacement mutants were inactive, and R192I was app roximately 30% as active as the wild-type, while the R197I mutant was equivalent to the mild-type control. A model is suggested for a unique binding motif in E. coli adenylyl cyclase in which there is a repetit ion of 3 basic residues on one face of a helix where there is an inter action with the three phosphate groups of ATP.