ADP-RIBOSYLATION OF ALPHA(I3)C20 BY THE S1 SUBUNIT AND DELETION PEPTIDES OF S1 OF PERTUSSIS TOXIN

Recombinant S1 subunit of PT (rS1) and two carboxyl-terminal deletion peptides, C180 and C204, which comprise the amino-terminal 180 and 204 amino acids of S1, respectively, were analyzed for the ability to ADP -ribosylate alpha(i3)C20, a synthetic peptide composed of the 20 carbo xyl-terminal amino acids of the a subunit of the heterotrimeric G prot ein G(i3). Under linear velocity conditions, C180 ADP-ribosylated alph a(i3)C20 at a 3-fold higher rate than either C204 or rS1. At variable NAD, rS1, C204, and C180 ADP-ribosylated alpha(i3)C20 with similar ini tial velocities which followed Michaelis-Menten kinetics. In contrast, at variable alpha(i3)C20, rS1, C204, and C180 ADP-ribosylated alpha(i 3)C20 with different initial velocities. At variable alpha(i3)C20, C20 4- and rS1-catalyzed ADP-ribosylation followed Michaelis-Menten kineti cs, while the velocity curve generated by C180 diverged from Michaelis -Menten kinetics. The rates of initial velocity of C180 did not fit th e Lineweaver-Burk equation, but could be transformed into the Hill equ ation which yielded a Hill coefficient of 2. This predicted that C180 possessed cooperativity between the two substrate binding sites. Other experiments showed that C180 ADP-ribosylated alpha(i3)C20 at 60% of t he rate for the ADP-ribosylation of G(t). These data showed that the e ntire catalytic mechanism for ADP-ribosylation resides within the firs t 180 amino acids of S1 and that the carboxyl-terminal 55 residues of S1 allow the ADP-ribosylation of alpha(i3)C20 to proceed via Michaelis -Menten kinetics. These data along with earlier studies (Krueger & Bar bieri, 1993) were also consistent with the presence of two G(t) protei n binding sites within S1. One G protein binding site was located with in the amino-tenninal 180 residues of S1 and interacted with a region of G(t alpha) defined by alpha(i3)C20. The second G protein binding si te was defined by residues 195-204 of S1 and was located within G(t al pha) but outside the alpha(i3)C20 binding site. This second interactio n provides a greater k(cat) and lower K-mGt for the ADP-ribosylation o f the heterotrimeric G(t) by S1.