Role of water in the catalytic cycle of E. coli dihydrofolate reductase

Abstract

Dihydrofolate reductase (DHFR) catalyzes the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of 7,8-dihydrofolate (H 2 F) to 5,6,7,8-tetrahydrofolate (H 4 F). Because of the absence of any ionizable group in the vicinity of N5 of dihydrofolate it has been proposed that N5 could be protonated directly by a water molecule at the active site in the ternary complex of the Escherichia coli enzyme with cofactor and substrate. However, in the X-ray structures representing the Michaelis complex of the E. coli enzyme, a water molecule has never been observed in a position that could allow protonation of N5. In fact, the side chain of Met 20 blocks access to N5. Energy minimization reported here revealed that water could be placed in hydrogen bonding distance of N5 with only minor conformational changes. The r.m.s. deviation between the conformation of the M20 loop observed in the crystal structures of the ternary complexes and the conformation adopted after energy minimization was only 0.79 Å. We performed molecular dynamics simulations to determine the accessibility by water of the active site of the Michaelis complex of DHFR. Water could access N5 relatively freely after an equilibration time of approximately 300 psec during which the side chain of Met 20 blocked water access. Protonation of N5 did not increase the accessibility by water. Surprisingly the number of near-attack conformations, in which the distance between the pro-R hydrogen of NADPH and C6 of dihydrofolate was less than 3.5 Å and the angle between C4 and the pro-R hydrogen of NADPH and C6 of dihydrofolate was greater than 120 degrees, did not increase after protonation. However, when the hydride was transferred from NADPH to C6 of dihydrofolate before protonation, the side chain of Met 20 moved away from N5 after approximately 100 psec thereby providing water access. The average time during which water was found in hydrogen bonding distance to N5 was significantly increased. These results suggest that hydride transfer might occur early to midway through the reaction followed by protonation. Such a mechanism is supported by the very close contact between C4 of NADP + and C6 of folate observed in the crystal structures of the ternary enzyme complexes, when the M20 loop is in its closed conformation.

Biosynthesis of Riboflavin: Structure and Properties of 2,5-Diamino-6-ribosylamino-4(3H)-pyrimidinone 5′-phosphate Reductase of Methanocaldococcus jannaschii

“…Although no water molecule could be observed in the X-ray structure representing the Michaelis complex of the E. coli enzyme, energy minimization calculations revealed that water could be placed in hydrogen bonding distance of N-5 with only minor conformational changes in DHFR. 49 No ionizable group in the vicinity of N-1′ of the modelled DAROPP could be found in the proposed active site of MjaRED. In anology to the DHFR reaction, a water molecule could also act as a direct proton donor of N-1′.…”

The Role of Arginine 28 in Catalysis by Dihydrofolate Reductase from the Hyperthermophile Thermotoga maritima

“…While extensive research has shown that the proton source in the EcDHFR‐catalysed reaction is most likely the solvent itself (see refs. 9, 15, 16, and references therein) this question has not been addressed in TmDHFR.…”

Searching Sequence Space: Two Different Approaches to Dihydrofolate Reductase Catalysis

“…[60] More recent molecular-dynamics (MD) calculations propose the reverse sequence. [61] Other factors have been proposed to facilitate catalysis. A positive electrostatic potential around the active site of Ec DHFR might steer binding of the negatively charged substrate and cofactor.…”