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J Kraut

Publications and source records attributed to J Kraut.

At least 19 recordsLinked to original sources
PubMed1997

Crystal structures of human DNA polymerase beta complexed with gapped and nicked DNA: evidence for an induced fit mechanism.

DNA polymerase beta (pol beta) fills single nucleotide (nt) gaps in DNA produced by the base excision repair pathway of mammalian cells. Crystal structures have been determined representing intermediates in the 1 nt gap-filling reaction of pol beta: the binary complex with a gapped DNA substrate (2.4 A resolution), the ternary complex including ddCTP (2.2 A), and the binary product complex containing only nicked DNA (2.6 A). Upon binding ddCTP to the binary gap complex, the thumb subdomain rotates into the closed conformation to contact the otherwise solvent-exposed ddCTP-template base pair. Thumb movement triggers further conformational changes which poise catalytic residue Asp192, dNTP, and template for nucleotidyl transfer, effectively assembling the active site. In the product nicked DNA complex, the thumb returns to the open conformation as in the gapped binary DNA complex, facilitating dissociation of the product. These findings suggest that pol beta may enhance fidelity by an induced fit mechanism in which correct base pairing between template and incoming dNTP induces alignment of catalytic groups for catalysis (via thumb closure), but incorrect base pairing will not. The structures also reveal that pol beta binds both gapped and nicked DNA with a 90 degrees kink occurring precisely at the 5'-phosphodiester linkage of the templating residue. If the DNA were not kinked in this way, contact between the thumb and dNTP-template base pair, presumably important for the checking mechanism, would be impossible, especially when the gap is but a single nucleotide. Such a 90 degrees kink may be a mechanistic feature employed by any polymerase involved in filling gaps to completion.

Catalysis↗
PubMed1997

Loop and subdomain movements in the mechanism of Escherichia coli dihydrofolate reductase: crystallographic evidence.

The reaction catalyzed by Escherichia coli dihydrofolate reductase (ecDHFR) cycles through five detectable kinetic intermediates: holoenzyme, Michaelis complex, ternary product complex, tetrahydrofolate (THF) binary complex, and THF.NADPH complex. Isomorphous crystal structures analogous to these five intermediates and to the transition state (as represented by the methotrexate-NADPH complex) have been used to assemble a 2.1 A resolution movie depicting loop and subdomain movements during the catalytic cycle (see Supporting Information). The structures suggest that the M20 loop is predominantly closed over the reactants in the holoenzyme, Michaelis, and transition state complexes. But, during the remainder of the cycle, when nicotinamide is not bound, the loop occludes (protrudes into) the nicotinamide-ribose binding pocket. Upon changing from the closed to the occluded conformation, the central portion of the loop rearranges from beta-sheet to 3(10) helix. The change may occur by way of an irregularly structured open loop conformation, which could transiently admit a water molecule into position to protonate N5 of dihydrofolate. From the Michaelis to the transition state analogue complex, rotation between two halves of ecDHFR, the adenosine binding subdomain and loop subdomain, closes the (p-aminobenzoyl)glutamate (pABG) binding crevice by approximately 0.5 A. Resulting enhancement of contacts with the pABG moiety may stabilize puckering at C6 of the pteridine ring in the transition state. The subdomain rotation is further adjusted by cofactor-induced movements (approximately 0.5 A) of helices B and C, producing a larger pABG cleft in the THF.NADPH analogue complex than in the THF analogue complex. Such movements may explain how THF release is assisted by NADPH binding. Subdomain rotation is not observed in vertebrate DHFR structures, but an analogous loop movement (residues 59-70) appears to similarly adjust the pABG cleft width, suggesting that these movements are important for catalysis. Loop movement, also unobserved in vertebrate DHFR structures, may preferentially weaken NADP+ vs NADPH binding in ecDHFR, an evolutionary adaptation to reduce product inhibition in the NADP+ rich environment of prokaryotes.

Crystallography, X-Ray↗
PubMed1997

pH-dependent conformational changes in Escherichia coli dihydrofolate reductase revealed by Raman difference spectroscopy.

The catalytic site of all dihydrofolate reductases contains an invariant carboxylic acid, equivalent to Asp-27 in Escherichia coli dihydrofolate reductase (ecDHFR). It has been found that various kinetic and ligand binding properties of ecDHFR show a pH profile with a pKa of about 6.5. The group responsible for this pKa is often assumed to be carboxyl group of Asp-27. To determine the ionization state of this carboxyl and its pKa, we have employed a novel method, based on Raman difference spectroscopy, to obtain its vibrational spectrum in situ. The method is general for the study of protein carboxyl groups, which are often significantly implicated in protein function and structure; this study establishes the method's limits and problems. The Raman difference spectrum between wild-type ecDHFR and the Asp-27 to serine mutant (D27S) in the pH range 5.6-9.0 has been taken. No protonation of the carboxyl group was detected, implying that its pKa is probably less than 5.0. We did, however, detect a pH dependence in the intensity of Raman bands in the difference spectrum with a pKa of 6.3, indicating that the apo enzyme undergoes a pH-dependent conformational change. Because the carboxyl group of Asp-27 at the active site is the only ionizable group in the binding site, other groups, away from the catalytic site, must be responsible for the pH behavior of ecDHFR.

Apoenzymes↗
PubMed1996

Crystal structures of human DNA polymerase beta complexed with DNA: implications for catalytic mechanism, processivity, and fidelity.

Mammalian DNA polymerase beta (pol beta) is a small (39 kDa) DNA gap-filling enzyme that comprises an amino-terminal 8-kDa domain and a carboxy-terminal 31-kDa domain. In the work reported here, crystal structures of human pol beta complexed with blunt-ended segments of DNA show that, although the crystals belong to a different space group, the DNA is nevertheless bound in the pol beta binding channel in the same way as the DNA in previously reported structures of rat pol beta complexed with a template-primer and ddCTP [Pelletier, H., Sawaya, M. R., Kumar, A., Wilson, S. H., & Kraut, J. (1994) Science 264, 1891-1903]. The 8-kDa domain is in one of three previously observed positions relative to the 31-kDa domain, suggesting that the 8-kDa domain may assume only a small number of stable conformations. The thumb subdomain is in a more open position in the human pol beta-DNA binary complex than it is in the rat pol beta-DNA-ddCTP ternary complex, and a closing thumb upon nucleotide binding could represent the rate-limiting conformational change that has been observed in pre-steady-state kinetic studies. Intermolecular contacts between the DNA and the 8-kDa domain of a symmetry-related pol beta molecule reveal a plausible binding site on the 8-kDa domain for the downstream oligonucleotide of a gapped-DNA substrate; in addition to a lysine-rich binding pocket that accommodates a 5'-PO4 end group, the 8-kDa domain also contains a newly discovered helix-hairpin-helix (HhH) motif that binds to DNA in the same way as does a structurally and sequentially homologous HhH motif in the 31-kDa domain. DNA binding by both HhH motifs is facilitated by a metal ion. In that HhH motifs have been identified in other DNA repair enzymes and DNA polymerases, the HhH-DNA interactions observed in pol beta may be applicable to a broad range of DNA binding proteins. The sequence similarity between the HhH motif of endonuclease III from Escherichia coli and the HhH motif of the 8-kDa domain of pol beta is particularly striking in that all of the conserved residues are clustered in one short sequence segment, LPGVGXK, where LPGV corresponds to a type II beta-turn (the hairpin turn), and GXK corresponds to a part of the HhH motif that is proposed to be critical for DNA binding and catalysis for both enzymes. These results suggest that endonuclease III and the 8-kDa domain of pol beta may employ a similar mode of DNA binding and may have similar catalytic mechanisms for their respective DNA lyase activities. A model for productive binding of pol beta to a gapped-DNA substrate requires a 90 degrees bend in the single-stranded template, which could enhance nucleotide selectivity during DNA repair or replication.

Amino Acid Sequence↗
PubMed1996

A structural basis for metal ion mutagenicity and nucleotide selectivity in human DNA polymerase beta.

When crystals of human DNA polymerase beta (pol beta) complexed with DNA [Pelletier, H., Sawaya, M. R., Wolfle, W., Wilson, S. H., & Kraut, J. (1996) Biochemistry 35, 12742-12761] are soaked in the presence of dATP and Mn2+, X-ray structural analysis shows that nucleotidyl transfer to the primer 3'-OH takes place directly in the crystals, even though the DNA is blunt-ended at the active site. Under similar crystal-soaking conditions, there is no evidence for a reaction when Mn2+ is replaced by Mg2+, which is thought to be the divalent metal ion utilized by most polymerases in vivo. These results suggest that one way Mn2+ may manifest its mutagenic effect on polymerases is by promoting greater reactivity than Mg2+ at the catalytic site, thereby allowing the nucleotidyl transfer reaction to take place with little or no regard to instructions from a template. Non-template-directed nucleotidyl transfer is also observed when pol beta-DNA cocrystals are soaked in the presence of dATP and Zn2+, but the reaction products differ in that the sugar moiety of the incorporated nucleotide appears distorted or otherwise cleaved, in agreement with reports that Zn2+ may act as a polymerase inhibitor rather than as a mutagen [Sirover, M. A., & Loeb, L. A. (1976) Science 194, 1434-1436]. Although no reaction is observed when crystals are soaked in the presence of dATP and other metal ions such as Ca2+, Co2+, Cr3+, or Ni2+, X-ray structural analyses show that these metal ions coordinate the triphosphate moiety of the nucleotide in a manner that differs from that observed with Mg2+. In addition, all metal ions tested, with the exception of Mg2+, promote a change in the side-chain position of aspartic acid 192, which is one of three highly conserved active-site carboxylate residues. Soaking experiments with nucleotides other than dATP (namely, dCTP, dGTP, dTTP, ATP, ddATP, ddCTP, AZT-TP, and dATP alpha S) reveal a non-base-specific binding site on pol beta for the triphosphate and sugar moieties of a nucleotide, suggesting a possible mechanism for nucleotide selectivity whereby triphosphate-sugar binding precedes a check for correct base pairing with the template.

Carcinogens↗
PubMed1996

Crystal structures of Escherichia coli dihydrofolate reductase complexed with 5-formyltetrahydrofolate (folinic acid) in two space groups: evidence for enolization of pteridine O4.

The crystal structure of Escherichia coli dihydrofolate reductase (ecDHFR, EC 1.5.1.3) as a binary complex with folinic acid (5-formyl-5,6,7,8-tetrahydrofolate; also called leucovorin or citrovorum factor) has been solved in two space groups, P6(1) and P6(5), with, respectively, two molecules and one molecule per asymmetric unit. The crystal structures have been refined to an R-factor of 14.2% at resolutions of 2.0 and 1.9 A. The P6(1) structure is isomorphous with several previously reported ecDHFR binary complexes [Bolin, J.T., Filman, D.J., Matthews, D.A., Hamlin, R.C., & Kraut, J. (1982) J. Biol. Chem. 257, 13650-13662; Reyes, V.M., Sawaya, M.R., Brown, K.A., & Kraut, J. (1995) Biochemistry 34, 2710-2723]; enzyme and ligand conformations are very similar to the P6(1) 5,10-dideazatetrahydrofolate complex. While the two enzyme subdomains of the P6(1) structure are nearly in the closed conformation, exemplified by the methotrexate P6(1) binary complex, in the P6(5) structure they are in an intermediate conformation, halfway between the closed and the fully open conformation of the apoenzyme [Bystroff, C., Oatley, S.J., & Kraut, J. (1990) Biochemistry 29, 3263-3277]. Thus crystal packing strongly influences this aspect of the enzyme structure. In contrast to the P6(1) structure, in which the Met-20 loop (residues 9-23) is turned away from the substrate binding pocket, in the P6(5) structure the Met-20 loop blocks the pocket and protrudes into the cofactor binding site. In this respect, the P6(5) structure is unique. Additionally, positioning of a Ca2+ ion (a component of the crystallization medium) is different in the two crystal packings: in the P6(1) structure it lies at the boundary between the two molecules of the asymmetric unit, while in P6(5) it coordinates two water molecules, the hydroxyl group of an ethanol molecule, and the backbone carbonyl oxygens of Glu-17, Asn-18, and Met-20. The Ca2+ ion thus stabilizes a single turn of 3(10) helix (residues 16-18 in the Met-20 loop), a second unique feature of the P6(5) crystal structure. The disposition of the N5-formyl group in these structures indicates formation, at least half of the time, of an intramolecular hydrogen bond between the formyl oxygen and O4 of the tetrahydropterin ring. This observation is consistent with the existence of an enol-keto equilibrium in which the enolic tautomer is favored when a hydrogen-bond acceptor is present between O4 and N5. Such would be the case whenever a water molecule occupies that site as part of a hypothetical proton-relay mechanism. Two arginine side chains, Arg-52 in the P6(5) structure and Arg-44 in molecule A of the P6(1) structure, are turned away drastically from the ligand (p-aminobenzoyl)glutamic acid moiety as compared with previously reported DHFR binary complex structures. As in the ecDHFR dideazatetrahydrofolate complex, in both the P6(1) and P6(5) structures a water molecule bridges pteridine O4 and Trp-22(N epsilon 1) with ideal geometry for hydrogen bonding, perhaps contributing to the slow release of 5,6,7,8-tetrahydrofolate from the enzyme-product complex. When either the P6(1) or the P6(5) structures are superimposed with the NADPH holoenzyme [Sawaya, M. R. (1994) Ph.D. Dissertation, University of California, San Diego], we find that the distances between the nicotinamide C4 and pteridine C6 and C7 are very short, 2.1 and 1.7 A in the P6(1) case and 2.0 and 1.4 A in the P6(5) case, perhaps in part explaining the more rapid release of tetrahydrofolate from the enzyme-product complex when NADPH is bound.

4-Aminobenzoic Acid↗
PubMed1996

Enzyme-DNA interactions required for efficient nucleotide incorporation and discrimination in human DNA polymerase beta.

In the crystal structure of a substrate complex, the side chains of residues Asn279, Tyr271, and Arg283 of DNA polymerase beta are within hydrogen bonding distance to the bases of the incoming deoxynucleoside 5'-triphosphate (dNTP), the terminal primer nucleotide, and the templating nucleotide, respectively (Pelletier, H., Sawaya, M. R., Kumar, A., Wilson, S. H., and Kraut, J. (1994) Science 264, 1891-1903). We have altered these side chains through individual site-directed mutagenesis. Each mutant protein was expressed in Escherichia coli and was soluble. The mutant enzymes were purified and characterized to probe their role in nucleotide discrimination and catalysis. A reversion assay was developed on a short (5 nucleotide) gapped DNA substrate containing an opal codon to assess the effect of the amino acid substitutions on fidelity. Substitution of the tyrosine at position 271 with phenylalanine or histidine did not influence catalytic efficiency (kcat/Km) or fidelity. The hydrogen bonding potential between the side chain of Asn279 and the incoming nucleotide was removed by replacing this residue with alanine or leucine. Although catalytic efficiency was reduced as much as 17-fold for these mutants, fidelity was not. In contrast, both catalytic efficiency and fidelity decreased dramatically for all mutants of Arg283 (Ala > Leu > Lys). The fidelity and catalytic efficiency of the alanine mutant of Arg283 decreased 160- and 5000-fold, respectively, relative to wild-type enzyme. Sequence analyses of the mutant DNA resulting from short gap-filling synthesis indicated that the types of base substitution errors produced by the wild-type and R283A mutant were similar and indicated misincorporations resulting in frequent T.dGTP and A.dGTP mispairing. With R283A, a dGMP was incorporated opposite a template thymidine as often as the correct nucleotide. The x-ray crystallographic structure of the alanine mutant of Arg283 verified the loss of the mutated side chain. Our results indicate that specific interactions between DNA polymerase beta and the template base, but not hydrogen bonding to the incoming dNTP or terminal primer nucleotide, are required for both high catalytic efficiency and nucleotide discrimination.

Base Sequence↗
PubMed1996

Identifying the physiological electron transfer site of cytochrome c peroxidase by structure-based engineering.

A technique was developed to evaluate whether electron transfer (ET) complexes formed in solution by the cloned cytochrome c peroxidase [CcP(MI)] and cytochromes c from yeast (yCc) and horse (hCc) are structurally similar to those seen in the respective crystal structures. Site-directed mutagenesis was used to convert the sole Cys of the parent enzyme (Cys 128) to Ala, and a Cys residue was introduced at position 193 of CcP(MI), the point of closest contact between CcP(MI) and yCc in the crystal structure. Cys 193 was then modified with a bulky sulfhydryl reagent, 3-(N-maleimidylpropionyl)-biocytin (MPB), to prevent yCc from binding at the site seen in the crystal. The MPB modification has no effect on overall enzyme structure but causes 20-100-fold decreases in transient and steady-state ET reaction rates with yCc. The MPB modification causes only 2-3-fold decreases in ET reaction rates with hCc, however. This differential effect is predicted by modeling studies based on the crystal structures and indicates that solution phase ET complexes closely resemble the crystalline complexes. The low rate of catalysis of the MPB-enzyme was constant for yCc in buffers of 20-160 mM ionic strength. This indicates that the low affinity complex formed between CcP(MI) and yCc at low ionic strength is not reactive in ET.

Animals↗
PubMed1995

Isomorphous crystal structures of Escherichia coli dihydrofolate reductase complexed with folate, 5-deazafolate, and 5,10-dideazatetrahydrofolate: mechanistic implications.

Crystal structures of Escherichia coli dihydrofolate reductase (ecDHFR, EC 1.5.1.3) in binary complexes with folate, 5-deazafolate (5dfol), and 5,10-dideazatetrahydrofolate (ddTHF) have been refined to R-factors of 13.7%, 14.9%, and 14.5%, respectively, all at 1.9 A. All three are isomorphous with a previously reported binary complex of ecDHFR with methotrexate (MTX), in space group P6(1), two molecules per asymmetric unit [Bolin, J. T., Filman, D. J., Matthews, D. A., Hamlin, R. C., & Kraut, J. (1982) J. Biol. Chem. 257, 13650-13662]. A hitherto unobserved water molecule is hydrogen bonded to the pteridine N5 and O4 in both molecules of the asymmetric unit of the folate complex (but not the 5dfol or ddTHF complexes), supporting the hypothesis that N5 protonation of bound substrate, an important step of the DHFR reaction, occurs by way of such a water molecule. There is no indication of a hydrogen bond between N8 of 5dfol and the backbone carbonyl of Ile-5, suggesting that the bacterial enzyme, unlike the human enzyme [Davies, J. F., II, Delcamp, T. J., Prendergast, N. J., Ashford, V. A., Freisheim, J. H., & Kraut, J. (1990) Biochemistry 29, 9467-9479], does not favor protonation at N8. Perhaps this explains why bacterial DHFR is much less effective than vertebrate DHFR in folate reduction. When the ecDHFR.NADPH complex (space group P3221; M. R. Sawaya, in preparation) is superimposed on the folate and 5dfol complexes, the distances from pteridine C6 to nicotinamide C4 were found to be 2.9 and 2.8 A, respectively, in close agreement with the theoretically calculated optimal distance in the transition state for hydride transfer [Wu, Y. D., & Houk, K. N. (1987) J. Am. Chem. Soc. 109, 906-908, 2226-2227]. In contrast to the planar ring system of folate or 5dfol, the reduced pteridine ring of ddTHF is severely puckered and bent toward the nicotinamide pocket, with the reduced pyridine ring assuming a half-chair type of conformation. This change in shape causes the pteridine ring to bind with O4 closer to Trp-22(N epsilon 1) by over 0.5 A, so that an invariant water molecule now bridges these two atoms with ideal hydrogen bonds. Furthermore, while the pABA rings of folate and 5dfol are nearly coincident and closer to the alpha C helix than to the alpha B helix, those of MTX and ddTHF are displaced along the binding crevice by approximately 1.1 and 0.6 A, respectively, and are equidistant from alpha B and alpha C.(ABSTRACT TRUNCATED AT 400 WORDS)

Binding Sites↗
PubMed1994

A cation binding motif stabilizes the compound I radical of cytochrome c peroxidase.

Cytochrome c peroxidase reacts with peroxide to form compound I, which contains an oxyferryl heme and an indolyl radical at Trp-191. The indolyl free radical has a half-life of several hours at room temperature, and this remarkable stability is essential for the catalytic function of cytochrome c peroxidase. To probe the protein environment that stabilizes the compound I radical, we used site-directed mutagenesis to replace Trp-191 with Gly or Gln. Crystal structures of these mutants revealed a monovalent cation binding site in the cavity formerly occupied by the side chain of Trp-191. Comparison of this site with those found in other known cation binding enzymes shows that the Trp-191 side chain resides in a consensus K+ binding site. Electrostatic potential calculations indicate that the cation binding site is created by partial negative charges at the backbone carbonyl oxygen atoms of residues 175 and 177, the carboxyl end of a long alpha-helix (residues 165-175), the heme propionates, and the carboxylate side chain of Asp-235. These features create a negative potential that envelops the side chain of Trp-191; the calculated free energy change for cation binding in this site is -27 kcal/mol (1 cal = 4.184J). This is more than sufficient to account for the stability of the Trp-191 radical, which our estimates suggest is stabilized by 7.8 kcal/mol relative to a Trp radical in solution.

Binding Sites↗
PubMed1994

Role of methionine 230 in intramolecular electron transfer between the oxyferryl heme and tryptophan 191 in cytochrome c peroxidase compound II.

The kinetics of electron transfer from cytochrome c (CC) to yeast cytochrome c peroxidase (CcP) compound I were studied by flash photolysis and stopped-flow spectroscopy. Flash photolysis studies employed horse CC derivatives labeled at specific lysine amino groups with (dicarboxybipyridine)bis-(bipyridine)ruthenium (Ru-CC). Initial electron transfer from Ru-CC reduced the indole radical on Trp-191 of CcP compound I [CMPI(IV,R.)], producing CMPII(IV,R). This reaction was biphasic for each of several Ru-CC derivatives, with rate constants which varied according to the position of the Ru label. For Ru-27-CC labeled at lysine 27, rate constants of 43,000 and 1600 s-1 were observed at pH 5.0 in 2 mM acetate. After reduction of the indole radical by Ru-CC, intramolecular electron transfer from Trp-191 to the oxyferryl heme in CMPII(IV,R) was observed, producing CMPII(III,R.). The rate constant and extent of this intramolecular electron transfer reaction were independent of both the protein concentration and the Ru-CC derivative employed. The rate constant decreased from 1100 s-1 at pH 5 to 550 s-1 at pH 6, while the extent of conversion of CMPII(IV,R) to CMPII(III,R.) decreased from 56% at pH 5 to 29% at pH 6. The reaction was not detected at pH 7.0 and above. The pH dependence of the rate and extent of this internal electron transfer reaction paralleled the pH dependence of the rate of bimolecular reduction of CMPII(IV,R) by native horse CC measured by stopped-flow spectroscopy at high ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Interaction domain for the reaction of cytochrome c with the radical and the oxyferryl heme in cytochrome c peroxidase compound I.

Site-directed mutants of cytochrome c peroxidase (CcP) were created to modify the interaction domain between CcP and yeast iso-1-cytochrome c (yCC) seen in the crystal structure of the CcP-yCC complex [Pelletier & Kraut (1992) Science 258, 1748-1755]. In the crystalline CcP-yCC complex, two acidic regions of CcP contact lysine residues on yCC. Mutants E32Q, D34N, E35Q, E290N, and E291Q were used to examine the effect of converting individual carboxylate side chains in the acidic regions to amides. The A193F mutant was used to test the effect of introducing a phenyl moiety at the point of closest contact between CcP and yCC in the crystal structure. Stopped-flow experiments carried out in 310 mM ionic strength buffer at pH 7 revealed that yCC initially reduced the indole radical on Trp-191 of the parent CcP compound I with a bimolecular rate constant ka = 2.5 x 10(8) M-1 s-1. A second molecule of yCC subsequently reduced the oxyferryl heme of compound II with a rate constant kb = 5 x 10(7) M-1 s-1. The bimolecular rate constants ka and kb were affected in parallel by each mutation examined. CcP mutants D34N and E290N that are closest to a complementary yCC lysine residue in the crystalline CcP-yCC complex gave the lowest values for ka and kb, which were 25-50% of the values of the CcP parent. Mutants E32Q and E291Q that are removed from the interaction domain gave the same ka and kb values as the CcP parent.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Crystal structure of rat DNA polymerase beta: evidence for a common polymerase mechanism.

Structures of the 31-kilodalton catalytic domain of rat DNA polymerase beta (pol beta) and the whole 39-kilodalton enzyme were determined at 2.3 and 3.6 angstrom resolution, respectively. The 31-kilodalton domain is composed of fingers, palm, and thumb subdomains arranged to form a DNA binding channel reminiscent of the polymerase domains of the Klenow fragment of Escherichia coli DNA polymerase I, HIV-1 reverse transcriptase, and bacteriophage T7 RNA polymerase. The amino-terminal 8-kilodalton domain is attached to the fingers subdomain by a flexible hinge. The two invariant aspartates found in all polymerase sequences and implicated in catalytic activity have the same geometric arrangement within structurally similar but topologically distinct palms, indicating that the polymerases have maintained, or possibly re-evolved, a common nucleotidyl transfer mechanism. The location of Mn2+ and deoxyadenosine triphosphate in pol beta confirms the role of the invariant aspartates in metal ion and deoxynucleoside triphosphate binding.

Animals↗
PubMed1994

Structures of ternary complexes of rat DNA polymerase beta, a DNA template-primer, and ddCTP.

Two ternary complexes of rat DNA polymerase beta (pol beta), a DNA template-primer, and dideoxycytidine triphosphate (ddCTP) have been determined at 2.9 A and 3.6 A resolution, respectively. ddCTP is the triphosphate of dideoxycytidine (ddC), a nucleoside analog that targets the reverse transcriptase of human immunodeficiency virus (HIV) and is at present used to treat AIDS. Although crystals of the two complexes belong to different space groups, the structures are similar, suggesting that the polymerase-DNA-ddCTP interactions are not affected by crystal packing forces. In the pol beta active site, the attacking 3'-OH of the elongating primer, the ddCTP phosphates, and two Mg2+ ions are all clustered around Asp190, Asp192, and Asp256. Two of these residues, Asp190 and Asp256, are present in the amino acid sequences of all polymerases so far studied and are also spatially similar in the four polymerases--the Klenow fragment of Escherichia coli DNA polymerase I, HIV-1 reverse transcriptase, T7 RNA polymerase, and rat DNA pol beta--whose crystal structures are now known. A two-metal ion mechanism is described for the nucleotidyl transfer reaction and may apply to all polymerases. In the ternary complex structures analyzed, pol beta binds to the DNA template-primer in a different manner from that recently proposed for other polymerase-DNA models.

Animals↗
PubMed1994

Determination by Raman spectroscopy of the pKa of N5 of dihydrofolate bound to dihydrofolate reductase: mechanistic implications.

Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate (H2folate) to tetrahydrofolate by NADPH, and this requires that the pteridine ring be protonated at N5. A long-standing puzzle has been how, at physiological pH, the enzyme can protonate N5 in view of its solution pKa of 2.6 and the fact that the only proton-donating group in the pterdine binding site, Asp-27, hydrogen bonds not to N5 but to the 2-amino group and N3 of the pterin ring. We have determined the pKa of N5 of dihydrofolate in the Escherichia coli DHFR/NADP+/H2folate ternary complex by Raman difference spectroscopy and found that the value is 6.5. In contrast, the pKa of N5 is less than 4.0 in either the binary complex, the ternary complex with an analogue of NADPH (H2NADPH), or the Asp27 to serine mutant DHFR (D27S) ternary complex with NADP+. Thus, one need not invoke proton donation from Asp-27 to N5 via a series of bound water molecules and/or pteridine-ring substituents. We propose instead that the N5 protonated form of H2folate is stabilized directly at the active site in the DHFR/NADPH/H2folate complex by specific interactions that form only in the ternary complex, involving perhaps a bound water molecule, the carboxamide moiety of the coenzyme, and/or the local electrostatic field of the enzyme molecule, to which an important contribution may be made by Asp-27.

Binding Sites↗
PubMed1994

Reaction of horse cytochrome c with the radical and the oxyferryl heme in cytochrome c peroxidase compound I.

The reactions of recombinant cytochrome c peroxidase [CcP(MI)] and a number of CcP(MI) mutants with native and ruthenium-labeled horse ferrocytochrome c have been studied by stopped-flow spectroscopy and laser flash photolysis. At 100 mM ionic strength, pH 7.5, native horse ferrocytochrome c reduces the radical on the indole group of Trp-191 in cytochrome c peroxidase compound I (CMPI) with a second-order rate constant of 1.3 x 10(8) M-1 s-1. Ferrocytochrome c then reduces the oxyferryl heme Fe(IV) in CMPII with a rate constant of 2.0 x 10(6) M-1 s-1. The rate constant for the reduction of the radical is nearly independent of pH from 5 to 8, but the rate constant for reduction of the oxyferryl heme Fe(IV) increases 33-fold as the pH is decreased from 8 to 5. This increase in rate is correlated with the pH dependence of the electron transfer equilibrium between the radical and the oxyferryl heme Fe(IV) in the transient form of CMPII. The second-order rate constants for reduction of the radical and the oxyferryl heme in the mutants Y39F, Y42F, H181G, W223F, and Y229F are nearly the same as for wild-type CcP(MI). The intracomplex rate constants for reduction of the radical in these mutants by the ruthenium-labeled cytochrome c derivatives are also similar to that for CcP(MI). This rules out a direct role for these aromatic residues in electron transfer.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

2.2 A structure of oxy-peroxidase as a model for the transient enzyme: peroxide complex.

The Fe+3-OOH complex of peroxidases has a very short half life, and its structure cannot be determined by conventional methods. The Fe+2-O2 complex provides a useful structural model for this intermediate, as it differs by only one electron and one proton from the transient Fe+3-OOH complex. We therefore determined the crystal structure of the Fe+2-O2 complex formed by a yeast cytochrome c peroxidase mutant with Trp 191 replaced by Phe. The refined structure shows that dioxygen can form a hydrogen bond with the conserved distal His residue, but not with the conserved distal Arg residue. When the transient Fe+3-OOH complex is modelled in a similar orientation, the active site of peroxidase appears to be optimized for catalysing proton transfer between the vicinal oxygen atoms of the peroxy-anion.

Binding Sites↗