Thiol reagents are substrates for the ADP‐ribosyltransferase activity of pertussis toxin

Abstract

Thiols such as cysteine and dithiothreitol are substrates for the ADP-ribosyltransferase activity of pertussis toxin. When cysteine was incubated with NAD ÷ and toxin at pH 7.5, a product containing ADP-ribose and cysteine (presumably ADP-ribosylcysteine) was isolated by high:performance liquid chromatography, and characterized by its composition and release of AMP with phosphodiesterase. Cysteine has a Km of 105 mM at saturating NAD + concentration. The ability of thiols to act as a substrate is one explanation for the very high concentrations (250 mM or greater) that have been observed to enhance the apparent NAD glycohydrolase activity of the toxin.Pertussis toxin; ADP-ribosylation; Thiol; NAD INTRODUCTIQN Pertussis toxin (review [1]) is a pathologicallyimportant protein secreted by Bordetella pertussis, the bacterium that causes whooping cough. The toxin is composed of five different subunits named S-1 to S-5 on the basis of decreasing molecular mass. The S-1 subunit, or A protomer, is an ADPribosyltransferase and also has NAD glycohydrolase activity. The remaining subunits constitute the B protomer, and are responsible for binding to a cell surface receptor and facilitating the entry of S-1 into the cell. Once within the cell, S-1 catalyses the transfer of ADP-ribose from NAD + to Gi, a GTP-binding regulatory protein of the adenylate cyclase complex. The toxin gene has been sequenced [2,3]. A Michaelis constant (Km) of 25/zM has been measured for NAD + in the NAD glycohydrolase reaction catalysed by the toxin, and generally . It purports to represent the breakdown of NAD + with water as the only acceptor for ADPribose. It has been found that this reaction is accelerated by sulphydryl compounds such as dithiothreitol, presumably because it is required for reducing a disulphide bond in the S-1 subunit, making it enzymically active [5]. However, the concentrations of thiol used have been much higher than is normally needed for activation (e.g. cholera toxin is analogous in many ways but needs only 10 mM at most). Concentrations of 250 mM dithiothreitol have often been used [4,6].Here, we show that thiol reagents are substrates for the ADP-ribosyltransferase reaction of the toxin, and that is why high concentrations accelerate the apparent glycohydrolase activity.

The purification of a cysteine-dependent NAD+ glycohydrolase activity from bovine erythrocytes and evidence that it exhibits a novel ADP-ribosyltransferase activity

“…This suggests that, although similar strategies were followed to purify these enzymes, different products were obtained. The affinity of the purified enzyme for cysteine was much higher than that exhibited by pertussis toxin (Km = 100 mM) [16], but similar to that shown by the human enzyme (Km = 4.4 mM) [21].…”

“…For cholera toxin [15] and pertussis toxin [16], it has been demonstrated that, in the absence of the protein target, the free amino acid may act as an acceptor for ADP-ribose. On the basis that endogenous enzymes may share the same enzyme mechanism as the bacterial toxins, this property was used to identify endogenous ADP-ribosyltransferases.…”

Mechanisms of Action of Nitrates

“…However, the effect of pertussis toxin may not be related to modification of a regulatory protein in the traditional manner. It has been shown that the site of ADP ribosylation of G proteins by pertussis toxin is a conserved cysteine residue located four amino acids from the COOH terminus of the (J.-subunit of the proteins [153], and thiol compounds may also function as ADP ribosylation substrates for pertussis toxin in vitro [156). The effect of pertussis toxin on relaxation and cGMP production might be due to inactivation of critical thiol groups necessary for the activation of soluble guanylate cyclase.…”

Pertussis toxin-catalyzed ADP-ribosylation of G0.alpha. with mutations at the carboxyl terminus

“…substrate (Lobban & van Heyningen, 1988). Proteolytic degradation at the carboxyl terminus, believed to result in the removal of the two terminal amino acids, appeared to inhibit ADP-ribosylation but not to affect association of a with fiy (Neer et al, 1988).…”