Conformation of an enzyme-bound substrate of staphylococcal nuclease as determined by NMR.
The dinucleoside phosphodiester dTdA is a slow substrate of staphylococcal nuclease (kcat = 3.8 X 10(-3) s-1) that forms binary E-S and ternary E-M-S complexes with Ca2+, Mn2+, Co2+, and La3+. The enzyme enhances the paramagnetic effects of Co2+ on 1/T1 and 1/T2 of the phosphorus and on 1/T1 of six proton resonances of dTdA, and these effects are abolished by binding of the competitive inhibitor 3',5'-pdTp. From paramagnetic effects of Co2+ on 1/T2 of phosphorus, koff of dTdA from the ternary E-Co(2+)-dTdA complex is greater than or equal to 4.8 X 10(4) s-1 and kon greater than or equal to 1.4 X 10(6) M-1 s-1, indicating the 1/T1 values to be in fast exchange. From paramagnetic effects of enzyme-bound Co2+ on 1/T1 of phosphorus and protons, with use of a correlation time of 1.6 ps on the basis of 1/T1 values at 250 and 600 MHz, 7 metal-nucleus distances and 9 lower-limit metal-nucleus distances are calculated. The long Co2+ to 31P distance of 4.1 +/- 0.9 A, which is intermediate between that expected for direct phosphoryl coordination (3.31 +/- 0.02 A) and a second sphere complex with an intervening water ligand (4.75 +/- 0.02 A), suggests either a distorted inner sphere complex or the rapid averaging of 18% inner sphere and 82% second sphere complexes and may explain the reduced catalytic activity with small dinucleotide substrates. Seventeen interproton distances and 108 lower limit interproton distances in dTdA in the ternary E-La(3+)-dTdA complex were determined by NOESY spectra at 50-, 100-, and 200-ms mixing times. While metal-substrate and interproton distances alone did not yield a unique structure, the combination of both sets of distances yielded a very narrow range of conformations for enzyme-bound dTdA, which was highly extended, with no base stacking, with high-anti glycosidic torsional angles for dT (64 degrees less than or equal to chi less than or equal to 73 degrees) and dA (66 degrees less than or equal to chi less than or equal to 68 degrees) and predominantly C-2'-endo sugar puckers for both nucleosides. Although the individual nucleosides are like those of B-DNA, their unstacked conformation, which is inappropriate for base pairing, as well as the conformational angles alpha and gamma of dA and zeta of dT, rule out B-DNA.(ABSTRACT TRUNCATED AT 400 WORDS)
Weber
D J
DJ
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Mullen
G P
GP
Mildvan
A S
AS
D002118:Calcium / Q000378:metabolism; D003035:Cobalt / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D015226:Dinucleoside Phosphates / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D007811:Lanthanum / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008345:Manganese / Q000378:metabolism; D008836:Micrococcal Nuclease / Q000378:metabolism*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D010758:Phosphorus / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013210:Staphylococcus / Q000201:enzymology
D004277:DNA, Single-Stranded; D015226:Dinucleoside Phosphates; D011994:Recombinant Proteins; C028145:2'-deoxythymidylyl-(3'-5')-2'-deoxyadenosine; D010758:Phosphorus; D003035:Cobalt; D008345:Manganese; D007811:Lanthanum; D008836:Micrococcal Nuclease; D002118:Calcium