Kinetic studies of yeast polyA polymerase indicate an induced fit mechanism for nucleotide specificity.
Polyadenylate polymerase (PAP) catalyzes the synthesis of 3'-polyadenylate tails onto mRNA. A comprehensive steady-state kinetic analysis of PAP was conducted which included initial velocity studies of the forward and reverse reactions, inhibition studies, and the use of alternative substrates. The reaction (A(n) + ATP <--> A(n+1) + PP(i)) is adequately described by a rapid equilibrium random mechanism. Several thermodynamic parameters for the reaction were determined or calculated, including the overall equilibrium constant (K(eq) = 84) and the apparent equilibrium constant of the internal step (K(int) = 4) which involves the rate-determining interconversion of central complexes. A large (100-fold) difference in Vmax accounts for nucleotide specificity (ATP vs CTP), despite an only 3-fold difference in Km. Comparison of the sulfur elemental effect on Vmax for ATP and CTP suggests that the chemical step is rate-determining for both reactions. Comparison of the sulfur elemental effect on Vmax/Km revealed differences in the mechanism by which either nucleotide is incorporated. Consistent with these data, an induced fit mechanism for nucleotide specificity is proposed whereby PAP couples a uniform binding mechanism, which selects for ATP, with a ground-state destabilization mechanism, which serves to accelerate the velocity for the correct substrate.
Balbo
Paul B
PB
Tufts University School of Medicine and Department of Biochemistry, Sackler School of Graduate Biomedical Sciences, 136 Harrison Avenue, Boston, Massachusetts 02111, USA.
Meinke
Gretchen
G
Bohm
Andrew
A
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D001667:Binding, Competitive; D003570:Cytidine Triphosphate / Q000737:chemistry; D011756:Diphosphates / Q000737:chemistry / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism; D004795:Enzyme Stability; D007700:Kinetics; D017616:Magnesium Compounds / Q000737:chemistry / Q000378:metabolism; D008956:Models, Chemical; D011061:Poly A / Q000737:chemistry / Q000378:metabolism; D026723:Polyadenylation; D011062:Polynucleotide Adenylyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity; D013455:Sulfur / Q000737:chemistry; D013816:Thermodynamics
D011756:Diphosphates; D004791:Enzyme Inhibitors; D017616:Magnesium Compounds; D029701:Saccharomyces cerevisiae Proteins; D011061:Poly A; D003570:Cytidine Triphosphate; D013455:Sulfur; D000255:Adenosine Triphosphate; C034732:magnesium pyrophosphate; C429559:PAP1 protein, S cerevisiae; D011062:Polynucleotide Adenylyltransferase