Bacteriophage T7 RNA polymerase and its active-site mutants. Kinetic, spectroscopic and calorimetric characterization.
It has been demonstrated that the amino acids Asp537, Asp812, Lys631, His811 and Tyr639 are involved in bacteriophage T7 RNA polymerase catalysis. In the present paper, we report kinetic, spectroscopic and calorimetric characterization of the wild-type and mutant T7 RNA polymerases generated at these five loci (D537N, E; K631M, R; Y639F, S, A, W; H811Q, A; D812N, E). The wild-type enzyme has a substantial amount of secondary structure as determined by CD analysis (alpha-helix, 43%; beta-sheet, 14%; beta-turn, 25%; unordered, 18%). The CD spectra of 12 mutants at five loci are very similar to that of the wild-type, except for the mutant Y639W. Within experimental error, the thermal transition temperatures measured by CD and DSC as well as the lambda max values of the fluorescence spectra were the same for the wild-type and all of the mutants. Therefore, the overall folding and stability of the mutant enzymes are very similar to those of the wild-type enzyme, although small local conformational changes cannot be excluded. For the synthesis of the pentamer pppGGACU, the mutants D537E and D812E showed an approximately two- to threefold decrease in (kcat)app and an approximately two- to threefold increase in (Km)app, relative to the wild-type, in contrast to the mutants D537N and D812N which exhibited no detectable activity. The mutant K631R showed a sevenfold reduction in (kcat)app and a two- to threefold increase in (Km)app, supporting our earlier observation with the mutant K631M that Lys631 may be involved in phosphodiester bond formation. The mutant Y639S can synthesize the trimer GGA with an approximately 50-fold decrease in (kcat)app and a tenfold increase in (Km)app, relative to the wild-type, underlining the importance of the phenyl ring of Tyr639. The mutant H811A, in which the side-chain at position 811 is incapable of forming a hydrogen bond, can synthesize the trimer GGA with an approximately tenfold decrease in (kcat)app and an approximately 35-fold increase in (Km)app. Thus, either the hydrogen-bonding capacity of this residue is non-essential or some other group can functionally substitute for the His811 side-chain. The wild-type enzyme showed significant effects of the base position in the sequence on the apparent binding constants for the NTPs. The kinetics of GpG-primed trimer, tetramer and pentamer synthesis on three 22 bp templates were investigated for the wild-type and mutant enzymes with measurable activity.(ABSTRACT TRUNCATED AT 400 WORDS)
Osumi-Davis
P A
PA
Department of Biochemistry, Colorado State University, Fort Collins 80523.
Sreerama
N
N
Volkin
D B
DB
Middaugh
C R
CR
Woody
R W
RW
Woody
A Y
AY
D000595:Amino Acid Sequence; D017123:Bacteriophage T7 / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013698:Templates, Genetic; D013816:Thermodynamics; D014158:Transcription, Genetic
D012321:DNA-Directed RNA Polymerases