title: Barnase has subsites that give rise to large rate enhancements.
abstract: Barnase is found to have a series of subsites for binding its substrates that confers large rate enhancements. Ribonucleotide substrates of the type Zp0Gp1Xp2Y have been synthesized, where p is phosphate, X, Y, and Z are nucleosides, and G is guanosine. G occupies the primary specificity site. The most important subsite is for p2, followed by that for Y. There appears to be no subsite for the Z or p0 positions. Occupation of the subsite for p2 gives rise to a 1000-fold increase in kcat/Km, composed of a 100-fold increase in kcat and a 10-fold decrease in Km. The Y subsite gives rise to further 20-fold increase in kcat/Km. Rates approaching diffusion control for kcat/Km are observed. kcat for the dinucleotide monophosphate GpU = 0.55 s-1, and Km = 240 microM; this compares with 53 s-1 and 20 microM for GpUp, and 3.3 x 10(3) s-1 and 17 microM for GpApA (the best substrate tested). Cleavage occurs at the 3'-phosphate of guanosine in all cases. There are differences in base specificity at the two subsites for X and Y downstream of the scissile bond. The binding energies of different substrates have been analyzed using thermodynamic cycles. These show that the contributions of the X and Y sites are nonadditive.
authors.affiliation: Department of Chemistry, University of Cambridge, England.
authors.forename: A G
authors.initials: AG
authors.lastname: Day
authors.forename: D
authors.initials: D
authors.lastname: Parsonage
authors.forename: S
authors.initials: S
authors.lastname: Ebel
authors.forename: T
authors.initials: T
authors.lastname: Brown
authors.forename: A R
authors.initials: AR
authors.lastname: Fersht
mesh_terms: D001407:Bacillus / Q000201:enzymology; D001426:Bacterial Proteins; D001665:Binding Sites; D007700:Kinetics; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D012260:Ribonucleases / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D009841:Oligonucleotides; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease