A Direct Pyrophosphatase-coupled Assay Provides New Insights into the Activation of the Secreted Adenylate Cyclase from Bordetella pertussis by Calmodulin

Abstract

Continuous recording of the activity of recombinant adenylate cyclase (CyaA) of Bordetella pertussis (EC 4.6.1.1) by conductimetric determination of enzyme-coupled pyrophosphate cleavage has enabled us to define a number of novel features of the activation of this enzyme by calmodulin and establish conditions under which valid activation data can be obtained. Activation either in the presence or absence of calcium is characterized by a concentration-dependent lag phase. The rate of formation and breakdown of the activated complex can be determined from an analysis of the lag phase kinetics and is in good agreement with thermodynamic data obtained by measuring the dependence of activation on calmodulin concentration, which show that calcium increases k on by about 30-fold. The rate of breakdown of the activated complex, formed either in the presence or absence of calcium, has been determined by dilution experiments and has been shown to be independent of the presence of calcium. The coupled assay is established as a rapid, convenient and safe method which should be readily applicable to the continuous assays of most other enzymes that catalyze reactions in which inorganic pyrophosphate is liberated.The secreted adenylate cyclase (CyaA) 1 of Bordetella pertussis is an important virulence factor and has a number of interesting structural and kinetic properties (1, 2). The N-terminal domain, containing the catalytic and calmodulin-binding domains, is joined to a large RTX (repeats in toxin) domain for membrane translocation (3, 4). Immunological evidence indicates that the catalytic domain may be of eukaryotic origin (5), but the calmodulin binding domain is bipartite and does not resemble classical calmodulin target sites very closely (6). CyaA, and the related edema factor from Bacillus anthracis, are among the most active adenylate cyclases yet characterized and show the largest known responses to calmodulin (4,7,8). CyaA is therefore ideal for evaluating new strategies for assay of adenylate cyclase and calmodulin and calmodulin antagonists (9). The fact that the catalytic reaction generates pyrophosphate as a second product has attracted little attention, but offers the possibility of assay by coupled detection of pyrophosphate cleavage; this could, in principle, enable continuous assays to be devised for many other important enzymes.Radiochemical assays for nucleotide cyclases (10, 11) tend to be used as single time point determinations, and due to logistics of isotope use, especially for 32 P, there is a tendency to run assays in large batches on collected samples. The aim here was to set up a simple rapid continuous recording method based on conductimetric determination of pyrophosphate cleavage (12) to be readily available for quality control and routine monitoring and which might be useful for assaying calmodulin and its inhibitors. However, the first application of the new method revealed time-dependent features of the kinetics for reactions initiated by addition of calmodulin and drew attention to proble...

Kinetic Studies of the Activation of Bordetella pertussis Adenylate Cyclase by Calmodulin

“…This general behavior was reported previously, but the investigators did not undertake a thorough kinetic analysis of their data.…”

Effect of Different Forms of Adenylate Cyclase Toxin ofBordetella pertussison Protection Afforded by an Acellular Pertussis Vaccine in a Murine Model

“…The purified proteins contained very low levels of endotoxin, at Ͻ0.1 endotoxin unit/g protein, as determined using a Kinetic-QCL (Biowhittaker) Limulus amoebocyte lysate assay, where 10 endotoxin units are equivalent to 1 ng of endotoxin (38). Only proCyaA and CyaA were enzymically active, with specific activities of 760 Ϯ 110 and 854 Ϯ 47 mol cAMP/min/mg protein, respectively, determined in the presence of 1 M calmodulin by using a rapid conductimetric procedure (22).…”

A novel non-radioactive primase–pyrophosphatase activity assay and its application to the discovery of inhibitors of Mycobacterium tuberculosis primase DnaG

“…These types of reactions include chromosomal DNA synthesis as well as the fatty acid adenylation, a critical biochemical step in fatty acid metabolism. In fact, inorganic pyrophosphatases from yeast and Escherichia coli were previously used in coupled assays with other enzymes, such as protein prenyltransferases (30), adenylate cyclase (31), acetyl-CoA synthetase (32) and aminoacyl-tRNA synthetase (33). In our assay, we use PPiase from Mtb (162 amino acid residues), which shares a modest (30%) amino acid residue sequence identity to its human counterpart, PPA1 (289 residues).…”