Crystal structures of nucleoside 2-deoxyribosyltransferase in native and ligand-bound forms reveal architecture of the active site.

BACKGROUND: Nucleoside 2-deoxyribosyltransferase plays an important role in the salvage pathway of nucleotide metabolism in certain organisms, catalyzing the cleavage of beta-2'-deoxyribonucleosides and the subsequent transfer of the deoxyribosyl moiety to an acceptor purine or pyrimidine base. The kinetics describe a ping-pong-bi-bi pathway involving the formation of a covalent enzyme-deoxyribose intermediate. The enzyme is produced by a limited number of microorganisms and its functions have been exploited in its use as a biocatalyst to synthesize nucleoside analogs of therapeutic interest. RESULTS: We describe the crystal structure of the enzyme with and without bound ligand. The native structure was solved by the single isomorphous replacement with anomalous scattering method (SIRAS) and refined to 2.5 A resolution resulting in a crystallographic R factor of 16.6%. The enzyme comprises a single domain that belongs to the general class of doubly-wound alpha/beta proteins; it also exhibits a unique nucleoside-binding motif. X-ray analysis of enzyme-purine and enzyme-pyrimidine complexes presented here reveals that the active site lies in a cleft formed by the edge of the beta sheet and two alpha helices and contains side chains from two subunits. CONCLUSIONS: These results indicate residues that may be important in substrate binding and catalysis and thus may serve as a framework for elucidating the mechanism of enzyme activity. In particular, the proposed nucleophile, Glu98, lies in the nucleoside-binding pocket at an appropriate position for nucleophilic attack. A comparison of the enzyme interactions with both a purine and pyrimidine ligand provides some insight into the structural basis for enzyme specificity.

Structural basis for the resilience of efavirenz (DMP-266) to drug resistance mutations in HIV-1 reverse transcriptase.

BACKGROUND: Efavirenz is a second-generation non-nucleoside inhibitor of HIV-1 reverse transcriptase (RT) that has recently been approved for use against HIV-1 infection. Compared with first-generation drugs such as nevirapine, efavirenz shows greater resilience to drug resistance mutations within HIV-1 RT. In order to understand the basis for this resilience at the molecular level and to help the design of further-improved anti-AIDS drugs, we have determined crystal structures of efavirenz and nevirapine with wild-type RT and the clinically important K103N mutant. RESULTS: The relatively compact efavirenz molecule binds, as expected, within the non-nucleoside inhibitor binding pocket of RT. There are significant rearrangements of the drug binding site within the mutant RT compared with the wild-type enzyme. These changes, which lead to the repositioning of the inhibitor, are not seen in the interaction with the first-generation drug nevirapine. CONCLUSIONS: The repositioning of efavirenz within the drug binding pocket of the mutant RT, together with conformational rearrangements in the protein, could represent a general mechanism whereby certain second-generation non-nucleoside inhibitors are able to reduce the effect of drug-resistance mutations on binding potency.

Characterization of a transforming N-ras gene in the human hepatoma cell line Hep G2: additional evidence for the importance of c-myc and ras cooperation in hepatocarcinogenesis.

The expression of the c-myc gene has previously been shown to be elevated and deregulated in the human hepatoma cell line Hep G2 (B. E. Huber and S. S. Thorgeirsson, Cancer Res., 47: 3414-3420, 1987). We now report that the Hep G2 N-ras gene is activated to a dominant-acting, transforming gene by a missense mutation in codon 61. Hep G2 DNA produced transformed foci when transfected into NIH 3T3 cells. Subsequent to a secondary round of transfection, Southern blot analysis of tumorigenic NIH 3T3 foci demonstrated the presence of human N-ras sequences. Nucleotide sequence analysis of one Hep G2 N-ras allele demonstrated that codons 12, 13, and 59 were normal and that codon 61 had a missense mutation (CAA to CTA). This mutation results in the incorporation of leucine instead of glutamine at residue 61 of the N-ras gene product, p21. N-ras sequences were amplified by the polymerase chain reaction from both Hep G2 genomic DNA and Hep G2 complementary DNA. Analysis of the amplified sequences demonstrated that only one Hep G2 N-ras allele exhibited the codon 61 mutation and that both the mutant and normal alleles were transcribed. Northern blot analysis demonstrated equivalent steady-state levels of N-ras transcripts in Hep G2 cells and normal human liver. The steady-state levels of N-ras and ornithine decarboxylase transcripts were positively correlated suggesting a positive relationship between N-ras expression and the replication rate of Hep G2 cells. c-Ki-ras and c-Ha-ras transcripts were not detected in either Hep G2 cells or normal human liver. Immunoprecipitation experiments using the monoclonal antibody Y13-259 demonstrated the presence of p21 in Hep G2 cells. Expression of a dominant-acting, transforming N-ras gene, in conjunction with the altered regulation of the c-myc gene, documents two important genetic lesions that could be responsible for the transformed phenotype of Hep G2 cells.

Functional testing of putative oligopeptide permease (Opp) proteins of Borrelia burgdorferi: a complementation model in opp(-) Escherichia coli.

Studies of the protein function of Borrelia burgdorferi have been limited by a lack of tools for manipulating borrelial DNA. We devised a system to study the function of a B. burgdorferi oligopeptide permease (Opp) orthologue by complementation with Escherichia coli Opp proteins. The Opp system of E. coli has been extensively studied and has well defined substrate specificities. The system is of interest in B. burgdorferi because analysis of its genome has revealed little identifiable machinery for synthesis or transport of amino acids and only a single intact peptide transporter operon. As such, peptide uptake may play a major role in nutrition for the organism. Substrate specificity for ABC peptide transporters in other organisms is determined by their substrate binding protein. The B. burgdorferi Opp operon differs from the E. coli Opp operon in that it has three separate substrate binding proteins, OppA-1, -2 and -3. In addition, B. burgdorferi has two OppA orthologues, OppA-4 and -5, encoded on separate plasmids. The substrate binding proteins interact with integral membrane proteins, OppB and OppC, to transport peptides into the cell. The process is driven by two ATP binding proteins, OppD and OppF. Using opp-deleted E. coli mutants, we transformed cells with B. burgdorferi oppA-1, -2, -4 or -5 and E. coli oppBCDF. All of the B. burgdorferi OppA proteins are able to complement E. coli OppBCDF to form a functional Opp transport system capable of transporting peptides for nutritional use. Although there is overlap in substrate specificities, the substrate specificities for B. burgdorferi OppAs are not identical to that of E. coli OppA. Transport of toxic peptides by B. burgdorferi grown in nutrient-rich medium parallels borrelial OppA substrate specificity in the complementation system. Use of this complementation system will pave the way for more detailed studies of B. burgdorferi peptide transport than currently available tools for manipulating borrelial DNA will allow.

Cytidine deaminase. The 2.3 A crystal structure of an enzyme: transition-state analog complex.

We have solved the structure of Escherichia coli cytidine deaminase (CDA) complexed to the transition state analog, 5-fluoroprimidin-2-one riboside. The monomer of the alpha 2 CDA dimer is composed of a small N-terminal alpha-helical domain with no obvious connection to the active sites, and two, larger, core domains. The two core domains have nearly identical tertiary structures and are related by approximate 2-fold symmetry, but lack internal amino acid sequence homology. Comparison of the core domain structure with known structures by sequence homology and structural compatibility searches suggests that the CDA tertiary structure cannot be superimposed on any known protein structure. The two active sites per dimer are formed across the subunit interface. The N-terminal core domain provides a pyrimidine nucleoside and zinc-binding pocket and the structurally homologous C-terminal core domain in the other monomer covers this active-site cleft, completely sequestering the ligand from solvent. The deeply buried zinc-binding site is formed by a novel "topological switch point" at the amino termini of two alpha-helices in consecutive alpha-beta-alpha-beta segments. The transition state analog is bound as a covalent hydrate at C4. The inhibitor hydroxyl oxygen atom interacts both with the zinc atom and the Glu104 carboxylate group, affording high differential affinity for the hydroxyl group relative to a hydrogen atom, in a manner reminiscent of that observed in adenosine deaminase (ADA). Unlike the latter enzyme, the zinc atom is coordinated in a tetrahedral ligand field to two cysteine and one histidine ligands, plus the hydroxyl group. Moreover, the inhibitor stereochemistry is of the opposite hand from that of the corresponding ADA inhibitor at C4(R), but is the same at the hydroxyl group O4(S). A consequence of these stereochemical differences is that in CDA a single conserved carboxylate side-chain, Glu104, can provide all of the necessary proton transfer functions involved in generating the zinc hydroxide nucleophile, and protonating the pyrimidine ring nitrogen atom and leaving amino group. The differences in zinc ligands, ligand-binding stereochemistry, and tertiary structures of CDA and ADA strongly suggest that the common features of transition state stabilization arose by convergent evolution.

Crystal structures of HIV-1 reverse transcriptases mutated at codons 100, 106 and 108 and mechanisms of resistance to non-nucleoside inhibitors.

Leu100Ile, Val106Ala and Val108Ile are mutations in HIV-1 reverse transcriptase (RT) that are observed in the clinic and give rise to resistance to certain non-nucleoside inhibitors (NNRTIs) including the first-generation drug nevirapine. In order to investigate structural mechanisms of resistance for different NNRTI classes we have determined six crystal structures of mutant RT-inhibitor complexes. Val108 does not have direct contact with nevirapine in wild-type RT and in the RT(Val108Ile) complex the biggest change observed is at the distally positioned Tyr181 which is > 8 A from the mutation site. Thus in contrast to most NNRTI resistance mutations RT(Val108Ile) appears to act via an indirect mechanism which in this case is through alterations of the ring stacking interactions of the drug particularly with Tyr181. Shifts in side-chain and inhibitor positions compared to wild-type RT are observed in complexes of nevirapine and the second-generation NNRTI UC-781 with RT(Leu100Ile) and RT(Val106Ala), leading to perturbations in inhibitor contacts with Tyr181 and Tyr188. Such perturbations are likely to be a factor contributing to the greater loss of binding for nevirapine compared to UC-781 as, in the former case, a larger proportion of binding energy is derived from aromatic ring stacking of the inhibitor with the tyrosine side-chains. The differing resistance profiles of first and second generation NNRTIs for other drug resistance mutations in RT may also be in part due to this indirect mechanism.

Studies on deo operon regulation in Escherichia coli: cloning and expression of the cytR structural gene.

The structural gene that encodes one repressor (the cytR-encoded repressor) of the Escherichia coli deo operon has been cloned from a lambda dmet transducing phage into the multicopy plasmid pBR322 by selecting for ApR, Lac- transformants of E. coli SS110(delta lac, cytR, tsx::lac). Restriction maps for the cytR+ plasmids have been generated and the position of the cytR gene on the cloned insert of these plasmids has been determined through deletion analysis. Results from maxicell experiments employing pCB001 and its cytR- derivatives suggest that the cytR gene encodes a protein with a subunit Mr of 37 000. In contrast to the complete repression of the deo operon obtained when deoR+ plasmids were introduced into E. coli SS201 (deoR, cytR), transformation of this DeoR-, CytR- strain with any of the cytR+ plasmids yields only clones which have phenotypes and Deo enzyme levels characteristic of a DeoR- single mutant. The data presented in this study are consistent with the interpretation that, in E. coli, the deoR-encoded repressor controls deo operon transcription initiating from both deo promoter-operator sites, PO1 and PO2. In contrast, the cytR-encoded repressor regulates deo operon expression only through deo promoter-operator site PO2.

Studies on deo operon regulation in Escherichia coli: cloning and expression of the deoR structural gene.

Recombinant plasmid pBR322 derivatives containing the Escherichia coli deoR structural gene (coding for one repressor of the deo operon) and a mutant allele of the cmlA gene (chromosomally encoded chloramphenicol resistance) have been constructed and the positions of these genes on a 6.3-kb EcoRI fragment have been determined. Transformation of an E. coli deoR single mutant with any of the deoR+ plasmids resulted in complementation of the chromosomal deoR mutation. More importantly, however, transformation of a deoR cytR double mutant with the deoR+ plasmids also resulted in complete repression of Deo enzyme synthesis. Based on these data, we conclude that transcription of the deo operon initiating from both the cAMP/CRP-independent promoter-operator site, PO1, and the cAMP/CRP-dependent promoter-operator site, PO2, is negatively controlled by the deoR-encoded repressor, whereas the cytR-encoded repressor regulates deo operon expression only from the cAMP/CRP-dependent promoter-operator site, PO2.

The cloning of the Escherichia coli K-12 deoxyribonucleoside operon.

A 6.1-kb EcoRI DNA fragment containing the four structural genes (deoC, deoA, deoB, deoD) of the deoxyribonucleoside operon has been cloned into the plasmid pMFS53. By use of a unique, asymmetrically positioned HindIII site on the 6.1 kb insert, plasmids containing the deoC,deoA genes (pMFS50) or the deoB,deoD genes (pMFS55) have been constructed. Enzyme assays performed on extracts prepared from clones harboring pMFS53, pMFS50 or pMFS55 revealed that each clone possessed amplified deo enzyme levels and that the spectrum of enzyme amplification corresponded to the genetic composition of the plasmids carried by each clone. A plasmid (pMFS50l) having functional deoA, deoB and deoD genes but devoid of the deo regulatory region and a portion of the deoC structural gene has been isolated following treatment of BamHI cleaved pMFS53 and BAL31 nuclease. Comparison of the deo enzyme levels for clones harboring pMFS53 and pMFS501 suggest that plasmid pMFS53 possesses a functional deo regulatory region in addition to the four structural genes of the operon.

Synthesis and antiviral activity of 2'-deoxy-4'-thio purine nucleosides.

A series of 2'-deoxy-4'-thioribo purine nucleosides was prepared by trans-N-deoxyribosylase-catalyzed reaction of 2'-deoxy-4'-thiouridine with a variety of purine bases. This synthetic procedure is an improvement over methods previously used to prepare purine 4'-thio nucleosides. The compounds were tested against hepatitis B virus (HBV), human cytomegalovirus (HCMV), herpes simplex virus (HSV-1 and HSV-2), varicella zoster virus (VZV), and human immunodeficiency virus (HIV-1). Cytotoxicity was determined in a number of cell lines. Several compounds were extremely potent against HBV and HCMV and had moderate to severe cytotoxicity in vitro. The lead compound from the series, 2-amino-6-(cyclopropylamino)purine 2'-deoxy-4'-thioriboside, was the most potent and selective agent against HCMV and HBV replication in vitro; however, this analogue was nephrotoxic when tested in vivo.

2-Amino-6-arylsulfonylbenzonitriles as non-nucleoside reverse transcriptase inhibitors of HIV-1.

A series of 2-amino-5-arylthiobenzonitriles (1) was found to be active against HIV-1. Structural modifications led to the sulfoxides (2) and sulfones (3). The sulfoxides generally showed antiviral activity against HIV-1 similar to that of 1. The sulfones, however, were the most potent series of analogues, a number having activity against HIV-1 in the nanomolar range. Structural-activity relationship (SAR) studies suggested that a meta substituent, particularly a meta methyl substituent, invariably increased antiviral activities. However, optimal antiviral activities were manifested by compounds where both meta groups in the arylsulfonyl moiety were substituted and one of the substituents was a methyl group. Such a disubstitution led to compounds 3v, 3w, 3x, and 3y having IC50 values against HIV-1 in the low nanomolar range. When gauged for their broad-spectrum antiviral activity against key non-nucleoside reverse transcriptase inhibitor (NNRTI) related mutants, all the di-meta-substituted sulfones 3u-z and the 2-naphthyl analogue 3ee generally showed single-digit nanomolar activity against the V106A and P236L strains and submicromolar to low nanomolar activity against strains E138K, V108I, and Y188C. However, they showed a lack of activity against the K103N and Y181C mutant viruses. The elucidation of the X-ray crystal structure of the complex of 3v (739W94) in HIV-1 reverse transcriptase showed an overlap in the binding domain when compared with the complex of nevirapine in HIV-1 reverse transcriptase. The X-ray structure allowed for the rationalization of SAR data and potencies of the compounds against the mutants.

Genetic analysis of Escherichia coli oligopeptide transport mutants.

The composition of the outer membrane channels formed by the OmpF and OmpC porins is important in peptide permeation, and elimination of these proteins from the Escherichia coli outer membrane results in a cell in which the primary means for peptide permeation through this cell structure has been lost. E. coli peptide transport mutants which harbor defects in genes other than the ompF/ompC genes have been isolated on the basis of their resistance to toxic tripeptides. The genetic defects carried by these oligopeptide permease-negative (Opp-) strains were found to map in two distinct chromosomal locations. One opp locus was trp linked and mapped to the interval between att phi 80 and galU. Complementation studies with F'123 opp derivatives indicated that this peptide transport locus resembles that characterized in Salmonella typhimurium as a tetracistronic operon (B. G. Hogarth and C. F. Higgins, J. Bacteriol. 153:1548-1551, 1983). The second opp locus, which we have designated oppE, was mapped to the interval between dnaC and hsd at 98.5 min on the E. coli chromosome. The differences in peptide utilization, sensitivity and resistance to toxic peptides, and the L-[U-14C]alanyl-L-alanyl-L-alanine transport properties observed with these Opp-E. coli strains demonstrated that the transport systems encoded by the trp-linked opp genes and by the oppE gene(s) have different substrate preferences. Mutants harboring defects in both peptide transport loci defined in this study would not grow on nutritional peptides except for tri-L-methionine, were totally resistant to toxic peptides, and would not actively transport L-[U-14C]alanyl-L-alanyl-L-alanine.

Amino acid substitutions in the CytR repressor which alter its capacity to regulate gene expression.

In Escherichia coli, transport and catabolism of nucleosides require expression of the genes composing the CytR regulon. Transcription initiation of cistrons in this gene family is activated by cyclic AMP-catabolite activator protein (cAMP-CAP), repressed by the CytR protein, and induced by cytidine. A random proofreading mutagenesis procedure and a genetic screen using udp-lac fusions have allowed the identification of distinct regions of the 341-amino-acid CytR polypeptide that are critical for repression of gene expression and response to induction. Determination of the ability of various CytR mutants to control gene expression in vivo indicated that the intrinsic affinity of the CytR protein for operator DNA is gene specific and that efficient repression of transcription by wild-type CytR is dependent on the interaction of CytR with cAMP-CAP. CytR mutants that were cytidine induction defective (CID) were characterized; these mutant proteins had only Asp-281 replaced. Data obtained with cytR delta M149, a dominant negative allele, indicated that the native CytR repressor is an oligomeric protein. Representative cytR mutations were combined with cytR delta M149, and the resulting hybrid repressors were tested for transdominance in a CytR+ E. coli strain. Amino acid substitutions A209E and C289Y suppressed the transdominance of CytR delta M149, suggesting that these replacements alter the normal protein contacts involved in repressor subunit-subunit association. In contrast, amino acid substitutions located in the N-terminal portion of the CytR protein had no effect on the transdominance of CytR delta M149. The results from this study suggest that the CytR repressor is an oligomeric, allosteric protein in which conformational changes are required for repression and derepression.

opp-lac Operon fusions and transcriptional regulation of the Escherichia coli trp-linked oligopeptide permease.

The transcriptional regulation of the Escherichia coli trp-linked opp operon that encodes the oligopeptide permease was investigated by using lambda plac Mu51-generated lac operon fusions. Synthesis of beta-galactosidase by strains harboring oppA-lac, oppB-lac, and oppD-lac fusions occurred at a basal level when the fusion-containing strains were grown in minimal medium. The addition of L-leucine or L-alanine to exponentially growing, aerobic cultures or shifting the aerobic fusion-containing strains to anaerobic growth medium increased the synthesis of beta-galactosidase from all opp-lac fusions. When transcription of the opp operon was induced by L-leucine, the differential rate of beta-galactosidase synthesis from each opp-lac fusion increased 8- to 10-fold; this increased rate of lacZ expression from the opp-lac fusions resulted in a 5- to 6-fold increase in total beta-galactosidase activity after maximum expression was achieved. Importantly, when F'123 derivatives harboring independently isolated E. coli opp-lac operon fusions were introduced into E. coli and Salmonella typhimurium, the data clearly demonstrated that the E. coli opp operon was expressed identically and responded to the same transcriptional regulatory signals in both E. coli and S. typhimurium. A comparison of beta-galactosidase synthesis by E. coli strains harboring an opp-lac operon fusion and either an oppE+ locus or an oppE mutation demonstrated that the reduction in peptide transport produced by the oppE mutation does not result from a decrease in the level of opp operon transcription.

Yeast orotidine-5'-phosphate decarboxylase: steady-state and pre-steady-state analysis of the kinetic mechanism of substrate decarboxylation.

The catalytically active form of monofunctional yeast orotidine-5'-phosphate decarboxylase was a dimer (E(2)). The dimer equilibrium dissociation constant was 0.25 microM in 0.01 M MOPS Na(+) at pH 7.2. The bimolecular rate constant for dimer formation was 1.56 microM(-1) s(-1). The dimeric form of the enzyme was stabilized by NaCl such that the enzyme was E(2) in 100 mM NaCl at all concentrations of enzyme tested. The kinetics of binding of OMP to E(2) was governed by two ionizations (pK(1) = 6.1 and pK(2) = 7.7). From studies with substrate analogues, the higher pK was assigned to a group on the enzyme that interacted with the pyrimidinyl moiety. The value of the lower pK was dependent on the substrate analogue, which suggested that it was not exclusively the result of ionization of the phosphoryl moiety. During the decarboxylation of OMP, the fluorescence of E(2) was quenched over 20%. The enzymatic species with reduced fluorescence was a catalytically competent intermediate that had kinetic properties consistent with it being the initial enzyme-substrate complex. The stoichiometry for binding of OMP to E(2) was one OMP per enzyme monomer. The value of the first-order rate constant for conversion of the enzyme-substrate complex to free enzyme (36 s(-1)) calculated from a single turnover experiment ([E] >> [S]) was slightly greater than the value of k(cat), 20 s(-1) (corrected for stoichiometry), calculated from steady-state data. In the single turnover experiments, the enzyme was E(2)*S, whereas in the steady-state turnover the experiment enzyme was E(2)*S(2). The similarity of these values suggested that the subunits were catalytically independent such that E(2)*S(2) could be treated as E*S and that conversion of the enzyme-substrate complex to E was k(cat). Kinetic data for the approach to the steady-state with OMP and E(2) yield a bimolecular association rate complex of 62 microM(-1) s(-1)and a dissociation rate constant for E*S of 60 s(-1). The commitment to catalysis was 0.25. By monitoring the effect of carbonic anhydrase on [H(+)] changes during a single turnover experiment, the initial product of the decarboxylation reaction was shown to be CO(2) not HCO(3-). UMP was released from the enzyme concomitantly with CO(2) during the conversion of E*S to E. Furthermore, the enzyme removed an enzyme equivalent of H(+) from solvent during this step of the reaction. The bimolecular rate constants for association of 6-AzaUMP and 8-AzaXMP, substrate analogues with markedly different nucleobases, had association rate constants of 112 and 130 microM(-1) s(-1), respectively. These results suggested that the nucleobase did not contribute significantly to the success of formation of the initial enzyme-substrate complex.

Characterization of cytR mutations that influence oligomerization of mutant repressor subunits.

In Escherichia coli, the transport and catabolism of nucleosides require expression of the genes composing the CytR regulon. The role of the CytR repressor in transcriptional regulation has been examined through a study of mutant CytR proteins. Two important and interrelated CytR mutants are encoded by cytR delta M149, a dominant negative allele, and cytRC289R. Studies with CytR delta M149 indicated that the native, repression-competent CytR protein is multimeric while the CytR amino acid substitution C-289-->R has been proposed to affect subunit oligomerization on the basis of its ability to suppress the transdominance of CytR delta M149. The present study identifies other CytR amino acid residues proximal to Cys-289 that may also participate in normal subunit oligomerization. Mutations in these CytR residues, cytRA307P, cytRM308R, and cytRL309P, encoded inactive repressors in a CytR- background and, when combined with cytR delta M149, yielded hybrid repressors that were recessive in a CytR+ genetic background. Because the stability and solubility observed for the new, mutant CytR proteins and the wild-type CytR protein were indistinguishable, these residue replacements, like the C-289-->R substitution, are envisaged as being located at the subunit interface and thus suppress the CytR delta M149 transdominance by blocking efficient and stable assembly of wild-type and hybrid CytR subunits. The assignment of CytR amino acids to a protein region involved in subunit association is also consistent with the observations that these CytR amino acids are roughly colinear with regions of the LacI repressor that influence monomer-dimer association and would be surface located by alignment to the E. coli galactose-binding protein crystal structure.

Nucleoside 2-deoxyribosyltransferase. Pre-steady-state kinetic analysis of native enzyme and mutant enzyme with an alanyl residue replacing Glu-98.

Nucleoside 2-deoxyribosyltransferase catalyzes cleavage of a 2'-deoxyribosylnucleoside (A) to a nucleobase (P) with deoxyribosylation of the enzyme. Substrates quenched the intrinsic fluorescence of native enzyme (E) and a catalytically inactive mutant enzyme (E98A enzyme). The time courses of these reactions were analyzed in terms of the following scheme where EX is the 2-deoxyribosyl ester of Glu-98. [formula: see text] The initial complexes between E and dAdo, dGuo, dIno, and dCyd or those between EX and the corresponding nucleobases were formed in a rapid equilibrium step. Native enzyme and E98A enzyme bound 2'-deoxyribosylnucleosides with similar affinities (k-1/k1). From a comparison of the time-dependent fluorescence changes associated with the reaction of native enzyme or E98A enzyme with these substrate, the kinetic step for 2-deoxyribosylation of Glu-98 was identified (k2 and k-2). dThd and dUrd quenched the fluorescence of native enzyme in a biphasic process. The late phase of this reaction was associated with 2-deoxyribosylation of Glu-98. The pre-steady-state kinetic constants calculated from fluorescence quenching data for dAdo and Cyt were consistent with the experimental values for the steady-state kinetic coefficients and the equilibrium constant of the reaction.