ADP-ribosylation of alpha i3C20 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 i3C20, a synthetic peptide composed of the 20 carboxyl-terminal amino acids of the alpha subunit of the heterotrimeric G protein Gi3. Under linear velocity conditions, C180 ADP-ribosylated alpha i3C20 at a 3-fold higher rate than either C204 or rS1. At variable NAD, rS1, C204, and C180 ADP-ribosylated alpha i3C20 with similar initial velocities which followed Michaelis-Menten kinetics. In contrast, at variable alpha i3C20, rS1, C204, and C180 ADP-ribosylated alpha i3C20 with different initial velocities. At variable alpha i3C20, C204- and rS1-catalyzed ADP-ribosylation followed Michaelis-Menten kinetics, while the velocity curve generated by C180 diverged from Michaelis-Menten kinetics. The rates of initial velocity of C180 did not fit the Lineweaver-Burk equation, but could be transformed into the Hill equation 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 i3C20 at 60% of the rate for the ADP-ribosylation of Gt. These data showed that the entire catalytic mechanism for ADP-ribosylation resides within the first 180 amino acids of S1 and that the carboxyl-terminal 55 residues of S1 allow the ADP-ribosylation of alpha i3C20 to proceed via Michaelis-Menten kinetics. These data along with earlier studies (Krueger & Barbieri, 1993) were also consistent with the presence of two Gt protein binding sites within S1.(ABSTRACT TRUNCATED AT 250 WORDS)
Finck-Barbançon
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V
Department of Microbiology, Medical College of Wisconsin, Milwaukee 53226.
Barbieri
J T
JT
D000595:Amino Acid Sequence; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020962:Heterotrimeric GTP-Binding Proteins; D007700:Kinetics; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D037342:Pertussis Toxin; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013696:Temperature; D010566:Virulence Factors, Bordetella / Q000737:chemistry / Q000378:metabolism*
D010446:Peptide Fragments; D010455:Peptides; D010566:Virulence Factors, Bordetella; C091653:alpha i3 C20; D009243:NAD; D011065:Poly(ADP-ribose) Polymerases; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D020962:Heterotrimeric GTP-Binding Proteins