Determination by Raman Spectroscopy of the pKa of N5 of Dihydrofolate Bound to Dihydrofolate Reductase: Mechanistic Implications

Chen, Y.-Q.; Kraut, Joseph; Blakley, Raymond L.; Callender, Robert

doi:10.1021/bi00189a001

Cited by 87 publications

(152 citation statements)

References 29 publications

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“…For Type I DHFR, it is generally thought that the protonation step precedes hydride transfer, thus creating a cationic intermediate which will more readily accept the hydride ion (45)(46)(47). Consistent with this view, vibrational spectroscopic studies have demonstrated that the N5 pK a of DHF is increased from 2.6 to 6.5 upon complex formation with E. coli DHFR (12)(13)(14)(15). In the type I enzyme, this protonation step is thought to involve an active site aspartyl (bacterial) or glutamyl (mammalian) residue.…”

Section: Catalytic Mechanismmentioning

confidence: 92%

“…These studies have shown that the pK a of N5 in the complex is < 4 (15), compared to the free pK a of 2.60 (35,52). Both these values compare with an N5 pK a value of 6.5 for DHF bound to the Type I enzyme from E. coli (12,13). These results demonstrate that elevation of the N5 pK a represents an important strategy for optimization of the catalytic rate constant for the chromosomal enzyme, while this perturbation apparently is not achieved for the Type II enzyme.…”

Section: Nih-pa Author Manuscriptmentioning

confidence: 97%

“…Therefore, the catalytic mechanism of Type II DHFR must differ significantly from that of the chromosomal DHFR (12,13,16,48,49) and also from the mechanism postulated for pteridine reductase (40). More specifically, there would appear to be no possibility of a keto-enol tautomeric equilibrium to facilitate N5 protonation, as has been proposed for the chromosomal DHFR (16).…”

Section: Catalytic Mechanismmentioning

confidence: 99%

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Crystal Structure of a Type II Dihydrofolate Reductase Catalytic Ternary Complex

et al. 2007

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Type II dihydrofolate reductase (DHFR) is a plasmid-encoded enzyme that confers resistance to bacterial DHFR-targeted antifolate drugs. It forms a symmetric homotetramer with a central pore which functions as the active site. Its unusual structure, which results in a promiscuous binding surface that accommodates either the Dihydrofolate (DHF) substrate or the NADPH cofactor, has constituted a significant limitation to efforts to understand its substrate specificity and reaction mechanism. We describe here the first structure of a ternary R67 DHFR•DHF•NADP + catalytic complex, resolved to1.26 Å. This structure provides the first clear picture of how this enzyme, which lacks the active site carboxyl residue that is ubiquitous in Type I DHFRs, is able to function. In the catalytic complex, the polar backbone atoms of two symmetry-related I68 residues provide recognition motifs that interact with the carboxamide on the nicotinamide ring, and the N3-O4 amide function on the pteridine. This set of interactions orients the aromatic rings of substrate and cofactor in a relative endo geometry in which the reactive centers are held in close proximity. Additionally, a central, hydrogen-bonded network consisting of two pairs of Y69-Q67-Q67′-Y69′ residues provides an unusually tight interface, which appears to serve as a "molecular clamp" holding the substrates in place in an orientation conducive to hydride transfer. In addition to providing the first clear insight regarding how this extremely unusual enzyme is able to function, the structure of the ternary complex provides general insights into how a mutationallychallenged enzyme, i.e., an enzyme whose evolution is restricted to four-residues-at-a-time active site mutations, overcomes this fundamental limitation. KeywordsR67 DHFR; Dihydrofolate Reductase; X-ray crystallography; ternary complex; Type II DHFR Antifolate drug therapy plays a critical role in the treatment of pathogenic and neoplastic diseases. The evolution of a plasmid-encoded, Type II dihydrofolate reductase (DHFR) provides one mechanism for bacterial evasion of drugs such as trimethoprim that target the bacterial dihydrofolate reductase enzyme (1-4). Type II DHFR is an extremely unusual enzyme that exhibits no apparent structural or evolutionary relationship with the type I (chromosomal) enzyme. It is one of the smallest enzymes known to self-assemble into an active quaternary structure, forming a homotetramer consisting of four 78-residue peptides * To whom correspondence should be addressed. This type of active site structure also creates substantial evolutionary and mutational challenges to the enzyme. Since each mutation will alter four active site residues at a time, most of the evolutionary pressure that would normally optimize enzyme function is compromised by the need to balance the effects of substitutions at all four symmetry-related sites. For example, a residue substitution on one monomer, which might promote folate N5 protonation, may also interfere with NADPH binding when it is prese...

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Section: Catalytic Mechanismmentioning

confidence: 92%

Section: Nih-pa Author Manuscriptmentioning

confidence: 97%

Section: Catalytic Mechanismmentioning

confidence: 99%

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Crystal Structure of a Type II Dihydrofolate Reductase Catalytic Ternary Complex

et al. 2007

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“…This assumes that the substrate carboxylate group and active-site side-chain functional groups are ionized, as found spectroscopically for functional groups in other enzymes (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18). The pyruvoyl dependent histidine decarboxylase does place its substrate carboxylate in a nonpolar environment (19), and nonpolar binding sites were successfully employed in the generation of PLP-dependent catalytic antibodies (20).…”

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confidence: 99%

Computational Studies on Nonenzymatic and Enzymatic Pyridoxal Phosphate Catalyzed Decarboxylations of 2-Aminoisobutyrate

Toney

2001

Biochemistry

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A computational study of nonenzymatic and enzymatic pyridoxal phosphate-catalyzed decarboxylation of 2-aminoisobutyrate (AIB) is presented. Four prototropic isomers of a model aldimine between AIB and 5′-deoxypyridoxal, with acetate interacting with the pyridine nitrogen, were employed in calculations of both gas phase and water model (PM3 and PM3-SM3) decarboxylation reaction paths. Calculations employing the transition state structures obtained for the four isomers allow the demonstration of stereoelectronic effects in transition state stabilization as well as a separation of the contributions of the Schiff base and pyridine ring moieties to this stabilization. The unprotonated Schiff base contribution (∼16 kcal/mol) is larger than that of the pyridine ring even when it is protonated (∼10 kcal/mol), providing an explanation of the catalytic power of pyruvoyl-dependent amino acid decarboxylases. An active site model of dialkylglycine decarboxylase was constructed and validated, and enzymatic decarboxylation reaction paths were calculated. The reaction coordinate is shown to be complex, with proton transfer from Lys272 to the coenzyme C4′ likely simultaneous with CR-CO 2 -bond cleavage. The proposed concerted decarboxylation/proton-transfer mechanism provides a simple explanation for the observed specificity of this enzyme toward oxidative decarboxylation.Pyridoxal phosphate (PLP) 1 dependent enzymes are ubiquitous and central to a wide variety of anabolic and catabolic pathways in the metabolism of nitrogen-containing compounds. The first, obligatory chemical step in all PLP dependent enzymatic reactions is formation of a Schiff base between the coenzyme aldehydic and substrate amino groups (Scheme 1). This common intermediate is termed the "external aldimine" in the literature. The general utility of PLP stems from its ability to stabilize carbanions formed adjacent to the Schiff base in the external aldimine intermediate through delocalization into the extended π bonded system (i.e., Schiff base and pyridine ring).Dunathan (32) proposed that the common fundamental mechanism that PLP dependent enzymes use to control reaction specificity is specific binding of the external aldimine intermediate such that the bond to be broken is oriented parallel to the p orbitals of the π-bonded system. This orientation maximizes orbital overlap between the nascent p orbital on CR and the π system in the transition state, thereby minimizing transition state energy. The magnitude of these proposed stereoelectronic effects have never been addressed.Dialkylglycine decarboxylase (DGD) is an intriguing PLP dependent enzyme that catalyzes two mechanistically different reactions: oxidative decarboxylation of 2,2-dialkylgycines in the first half-reaction, and transamination of R-keto acids in the second. Several DGD structures have been solved by X-ray crystallography (1-4).The active site structure shows that, to be bound productively for decarboxylation, a carboxylate group would make multiple favorable hydrogen bonding an...

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“…Moreover, this conclusion agreed well with pH-dependent kinetic and mutagenesis studies of the E. coli enzyme (4, 13, 14). A protein-induced "preprotonation" of N5 goes a long way toward understanding the catalytic mechanism because N5 must protonate along the pathway and also ab initio calculations suggest that protonation will facilitate hydride transfer (10,15,16). On the other hand, difference Raman studies of dihydroneopterin aldolase, another enzyme in the folate synthesis pathway whose structural and chemical similarities to E. coli DHFR prompted the suggestion of a reaction mechanism similar to that of chromosomal DHFRs (17), found that N5 is unprotonated above pH 6 (18).…”

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confidence: 99%

Vibrational Structure of Dihydrofolate Bound to R67 Dihydrofolate Reductase

Deng¹,

Callender²,

Howell³

2001

Journal of Biological Chemistry

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R67 is a Type II dihydrofolate reductase (DHFR) that catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate by facilitating the addition of a proton to N5 of DHF and the transfer of a hydride ion from NADPH to C6. Because this enzyme is a plasmid-encoded DHFR from trimethoprim-resistant bacteria, extensive studies on R67 with various methods have been performed to elucidate its reaction mechanism. Here, Raman difference measurements, conducted on the ternary complex of R67⅐NADP؉ ⅐DHF believed to be an accurate mimic of the productive DHFR⅐NADPH⅐DHF complex, show that the pK a of N5 in the complex is less than 4. This is in clear contrast to the behavior observed in Escherichia coli DHFR, a substantially more efficient enzyme, where the pK a of bound DHF at N5 is increased to 6.5 compared with its solution value of 2.6. A comparison of the ternary complexes in R67 and E. coli DHFRs suggests that enzymic raising of the pK a at N5 can significantly increase the catalytic efficiency of the hydride transfer step. However, R67 shows that even without such a strategy an effective DHFR can still be designed.Dihydrofolate reductase (5,6,7, EC 1.5.1.3, DHFR) 1 catalyzes the reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate (THF) by facilitating the addition of a proton to N5 of DHF and the transfer of a hydride ion from NADPH to C6 (Scheme I). DHFR is an important enzyme as it is required for the production of purines, thymidylate, and a few amino acids; therefore, it has been the target of antimicrobial drugs for a long time. Various drugresistant types of DFHR have been discovered, and it has been found that Type II DHFRs are particularly interesting as they are genetically unrelated to chromosomal DHFRs. One Type II DHFR, R67, is a homotetramer, each monomer consisting of 78 amino acids. Crystallographic studies of R67 show a very different structure compared with chromosomal DHFR, either at the level of the overall protein fold or at the active site (1, 2). Yet R67 compares quite well as an enzyme; its k cat ϭ 1.3 s Ϫ1 (3) can be compared with a hydride transfer rate of 238 s Ϫ1 for chromosomal DHFR at pH 7 (4, 5). Hence, it is interesting to compare the catalytic mechanism of R67 with chromosomal DHFRs (2).Extensive kinetic, site-directed mutagenesis, x-ray crystallographic, and theoretical molecular modeling studies have been performed on Escherichia coli chromosomal DHFR to elucidate its reaction mechanism (6 -9). The electronic nature of the ground state within the active site in the productive DHFR⅐NADPH⅐DHF complex is unclear, and this is important to an understanding of the reaction mechanism of DHFR. A key issue has to do with whether or not N5 of the pteridine ring of DHF is protonated in the ground state in the DHFR⅐NADPH⅐DHF complex and hence precedes hydride transfer or occurs later in the reaction pathway. A recent study using Raman difference spectroscopy of the E. coli DHFR⅐NADP ϩ ⅐DHF complex, believed to be an accurate mimic of the productive DHFR⅐NADPH⅐DHF complex, identi...

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Determination by Raman Spectroscopy of the pKa of N5 of Dihydrofolate Bound to Dihydrofolate Reductase: Mechanistic Implications

Cited by 87 publications

References 29 publications

Crystal Structure of a Type II Dihydrofolate Reductase Catalytic Ternary Complex

Crystal Structure of a Type II Dihydrofolate Reductase Catalytic Ternary Complex

Computational Studies on Nonenzymatic and Enzymatic Pyridoxal Phosphate Catalyzed Decarboxylations of 2-Aminoisobutyrate

Vibrational Structure of Dihydrofolate Bound to R67 Dihydrofolate Reductase

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