THE COMPLEX BETWEEN RIBONUCLEASE-T1 AND 3'GMP SUGGESTS GEOMETRY OF ENZYMATIC-REACTION PATH - AN X-RAY STUDY

The crystal structure of the complex between ribonuclease Tl and 3'GMP suggests that (a) a substrate GpN is bound to the active site of ribonuclease Tl in a conformation that actively supports the catalytic process, (b) the reaction occurs in an in-line process, (c) His40 NepsilonH+ activates 02'-H, (d) Glu58 carboxylate acts as base and His92 NepsilonH+ as acid in a general acid-base catalysis. The crystals have the monoclinic space group P2(1), a = 4.968 nm, b = 4.833 nm, c = 4.048 nm, beta = 90.62-degrees with two molecules in the asymmetric unit. The structure was determined by molecular replacement and refined to R = 15.3% with 11338 data greater-than-or-equal-to 1sigma(F(o)) in the resolution range 1.0-0.2 nm; this includes 180 water molecules and two Ca2+. The structure of ribonuclease T1 is as previously observed. 3'GMP is bound in syn conformation; guanine is located in the specific recognition site, the ribose adopts C4'-exo puckering, the ribose phosphate is extended with torsion angle epsilon in trans. The 02'-H group is activated by accepting and donating hydrogen bonds from His40 NepsilonH+ and to Glu58 Oepsilon1; the phosphate is hydrogen bonded to Glu58 Oepsilon2H, Arg77 NespilonH+ and Neta2H+, Tyr38 0etaH, His92 NepsilonH+. The conformation of ribose phosphate is such that 02' is at a distance of 0.31 nm from phosphorus, and opposite the P-OP3 bond which accepts a hydrogen bond from His92 NepsilonH+; we infer from a model building study that this bond is equivalent to the scissile P-05' in a substrate GpN.