Biomedical subjects

K S Anderson

Publications and source records attributed to K S Anderson.

At least 19 recordsLinked to original sources

PubMed2001

Equivalent induction of telomerase-specific cytotoxic T lymphocytes from tumor-bearing patients and healthy individuals.

Although high frequencies of T lymphocytes specific for certain tumor-associated antigens have been detected in some cancer patients, increasing evidence suggests that these T cells may be functionally defective in vivo and fail to induce meaningful clinical responses. One strategy to overcome this limitation is to target novel antigens that are ignored during the natural antitumor immune response but are nevertheless capable of triggering effector T-cell responses against tumors after optimal presentation by antigen-presenting cells. Here, we show that the telomerase catalytic subunit (hTERT)-a nearly universal tumor antigen identified by epitope deduction rather than from patient immune responses-is immunologically ignored by patients despite progressive tumor burden. Nevertheless, HLA-A2-restricted CTLs against hTERT are equivalently induced ex vivo from patients and healthy individuals and efficiently kill human tumor cell lines and primary tumors. Thus, telomerase-specific T cells from cancer patients are spared functional inactivation because of immunological ignorance. These findings support clinical efforts to target the hTERT as a tumor antigen with broad therapeutic potential.

Adult↗

PubMed2001

Toxicity of antiviral nucleoside analogs and the human mitochondrial DNA polymerase.

To examine the role of the mitochondrial polymerase (Pol gamma) in clinically observed toxicity of nucleoside analogs used to treat AIDS, we examined the kinetics of incorporation catalyzed by Pol gamma for each Food and Drug Administration-approved analog plus 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil (FIAU), beta-L-(-)-2',3'-dideoxy-3'-thiacytidine (-)3TC, and (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA). We used recombinant exonuclease-deficient (E200A), reconstituted human Pol gamma holoenzyme in single turnover kinetic studies to measure K(d) (K(m)) and k(pol) (k(cat)) to estimate the specificity constant (k(cat)/K(m)) for each nucleoside analog triphosphate. The specificity constants vary more than 500,000-fold for the series ddC > ddA (ddI) > 2',3'-didehydro-2',3'-dideoxythymidine (d4T) >> (+)3TC >> (-)3TC > PMPA > azidothymidine (AZT) >> Carbovir (CBV). Abacavir (prodrug of CBV) and PMPA are two new drugs that are expected to be least toxic. Notably, the higher toxicities of d4T, ddC, and ddA arose from their 13-36-fold tighter binding relative to the normal dNTP even though their rates of incorporation were comparable with PMPA and AZT. We also examined the rate of exonuclease removal of each analog after incorporation. The rates varied from 0.06 to 0.0004 s(-1) for the series FIAU > (+)3TC approximately equal to (-)3TC > CBV > AZT > PMPA approximately equal to d4T >> ddA (ddI) >> ddC. Removal of ddC was too slow to measure (<0.00002 s(-1)). The high toxicity of dideoxy compounds, ddC and ddI (metabolized to ddA), may be a combination of high rates of incorporation and ineffective exonuclease removal. Conversely, the more effective excision of (-)3TC, CBV, and AZT may contribute to lower toxicity. FIAU is readily extended by the next correct base pair (0.13 s(-1)) faster than it is removed (0.06 s(-1)) and, therefore, is stably incorporated and highly mutagenic. We define a toxicity index for chain terminators to account for relative rates of incorporation versus removal. These results provide a method to rapidly screen new analogs for potential toxicity.

Base Sequence↗

PubMed2001

Insights into the molecular mechanism of mitochondrial toxicity by AIDS drugs.

Several of the nucleoside analogs used in the treatment of AIDS exhibit a delayed clinical toxicity limiting their usefulness. The toxicity of nucleoside analogs may be related to their effects on the human mitochondrial DNA polymerase (Pol gamma), the polymerase responsible for mitochondrial DNA replication. Among the AIDS drugs approved by the FDA for clinical use, two are modified cytosine analogs, Zalcitabine (2',3'-dideoxycytidine (ddC)) and Lamivudine (beta-d-(+)-2',3'-dideoxy-3'-thiacytidine ((-)3TC])). (-)3TC is the only analog containing an unnatural l(-) nucleoside configuration and is well tolerated by patients even after long term administration. In cell culture (-)3TC is less toxic than its d(+) isomer, (+)3TC, containing the natural nucleoside configuration, and both are considerably less toxic than ddC. We have investigated the mechanistic basis for the differential toxicity of these three cytosine analogs by comparing the effects of dideoxy-CTP), (+)3TC-triphosphate (TP), and (-)3TC-TP on the polymerase and exonuclease activities of recombinant human Pol gamma. This analysis reveals that Pol gamma incorporates (-)3TC-triphosphate 16-fold less efficiently than the corresponding (+)isomer and 1140-fold less efficiently than dideoxy-CTP, showing a good correlation between incorporation rate and toxicity. The rates of excision of the incorporated analogs from the chain-terminated 3'-end of the DNA primer by the 3'-5'-exonuclease activity of Pol gamma were similar (0.01 s(-)1) for both 3TC analogs. In marked contrast, the rate of exonuclease removal of a ddC chain-terminated DNA occurs at least 2 orders of magnitude slower, suggesting that the failure of the exonuclease to remove ddC may play a major role in its greater toxicity. This study demonstrates that direct analysis of the mitochondrial DNA polymerase structure/function relationships may provide valuable insights leading to the design of less toxic inhibitors.

Acquired Immunodeficiency Syndrome↗

PubMed2001

Y265H mutator mutant of DNA polymerase beta. Proper teometric alignment is critical for fidelity.

DNA polymerases have the unique ability to select a specific deoxynucleoside triphosphate from a pool of similarly structured substrates. One of these enzymes, DNA polymerase beta, offers a simple system to relate polymerase structure to the fidelity of DNA synthesis. In this study, a mutator DNA polymerase beta, Y265H, was identified using an in vivo genetic screen. Purified Y265H produced errors at a 40-fold higher frequency than the wild-type protein in a forward mutation assay. At 37 degrees C, transient kinetic analysis demonstrated that the alteration caused a 111-fold decrease in the maximum rate of polymerization and a 117-fold loss in fidelity for G misincorporation opposite template A. Our data suggest that the maximum rate of polymerization was reduced, because Y265H was dramatically impaired in its ability to perform nucleotidyl transfer in the presence of the correct nucleotide substrate. In contrast, at 20 degrees C, the mutant protein had a fidelity similar to wild-type enzyme. Both proteins at 20 degrees C demonstrate a rapid change in protein conformation, followed by a slow chemical step. These data suggest that proper geometric alignment of template, 3'-OH of the primer, magnesium ions, dNTP substrates, and the active site residues of DNA polymerase beta are important factors in polymerase fidelity and provide the first evidence that Tyr-265 is important for this alignment to occur properly in DNA polymerase beta.

Bacterial Proteins↗

PubMed2001

Tumour immunotherapy: new tools, new treatment modalities and new T-cell antigens.

Tumour immunology has seen many exciting developments in the last few years. In addition to tumour antigens that are defined by antitumour T- and B-cell responses in patients, the human telomerase reverse transcriptase has been identified by 'reverse immunology' as the first truly universal tumour antigen. Molecular remission has been associated with a cancer vaccine that targets the clonal idiotype of B-cell malignancies, and sophisticated cellular vaccines (including fusions of tumour cells and antigen-presenting cells) have demonstrated promising results. Moreover, our capabilities of measuring immunity have been significantly enhanced by novel technology, such as major histocompatibility complex (MHC)-peptide tetramers and ELISPOT analysis. We are now capable of tracking antigen-specific T cells at a single cell level. This review will analyse recent developments and highlight some important issues that need to be addressed in the future.

Antigens, Neoplasm↗

PubMed2001

Deoxythioguanosine triphosphate impairs HIV replication: a new mechanism for an old drug.

Inhibition of HIV-1 reverse transcriptase (RT) and HIV protease are effective mechanisms for anti-retroviral agents, and the combined use of mechanistically different medications has markedly improved the treatment of HIV infected patients. The active metabolite of mercaptopurine and thioguanine (TG), deoxythioguanosine triphosphate, was shown to be incorporated into DNA by the polymerase function of HIV-1 RT and then to abrogate RNA cleavage by HIV-1 RNaseH. Treatment of human lymphocyte cultures with thioguanine produced substantial inhibition of HIV replication (IC(50)=0.035 microM, IC(95)=15.4 microM), with minimal toxicity to host lymphocytes (<10% at 15.4 microM TG, P<0.000005). Furthermore, low concentrations of TG and zidovudine were synergistic in inhibiting HIV replication in human lymphocytes (synergy volume=19 microM(2)%), without additive cytotoxicity to host lymphocytes. Thus, thiopurines are novel anti-retroviral agents that alter the DNA-RNA substrates for HIV RNaseH, thereby abrogating early stages of HIV replication.

Anti-HIV Agents↗

PubMed2001

Mechanism of action of 1-beta-D-2,6-diaminopurine dioxolane, a prodrug of the human immunodeficiency virus type 1 inhibitor 1-beta-D-dioxolane guanosine.

(-)-beta-D-2,6-Diaminopurine dioxolane (DAPD), is a nucleoside reverse transcriptase (RT) inhibitor with activity against human immunodeficiency virus type 1 (HIV-1). DAPD, which was designed as a water-soluble prodrug, is deaminated by adenosine deaminase to give (-)-beta-D-dioxolane guanine (DXG). By using calf adenosine deaminase a K(m) value of 15 +/- 0.7 microM was determined for DAPD, which was similar to the K(m) value for adenosine. However, the k(cat) for DAPD was 540-fold slower than the k(cat) for adenosine. In CEM cells and peripheral blood mononuclear cells exposed to DAPD or DXG, only the 5'-triphosphate of DXG (DXG-TP) was detected. DXG-TP is a potent alternative substrate inhibitor of HIV-1 RT. Rapid transient kinetic studies show the efficiency of incorporation for DXG-TP to be lower than that measured for the natural substrate, 2'-deoxyguanosine 5'-triphosphate. DXG-TP is a weak inhibitor of human DNA polymerases alpha and beta. Against the large subunit of human DNA polymerase gamma a K(i) value of 4.3 +/- 0.4 microM was determined for DXG-TP. DXG showed little or no cytotoxicity and no mitochondrial toxicity at the concentrations tested.

Adenosine Deaminase Inhibitors↗

PubMed2001

Linking genomics to immunotherapy by reverse immunology--'immunomics' in the new millennium.

The disclosure of the human genome sequence and rapid advances in genomic expression profiling have revolutionized our knowledge about molecular changes in malignant diseases. Rapidly growing gene expression databases and improvements in bioinformatics tools set the stage for new approaches using large-scale molecular information to develop specific therapeutics in cancer. On one hand, the ability to detect clusters of genes differentially expressed in normal and malignant tissue may lead to widely applicable targeting of defined molecular structures. On the other hand, analyzing the 'molecular fingerprint' of an individual tumor raises the possibility of developing customized therapeutics. One approach to use the emerging new datasets for the development of novel therapeutics is to identify genes that are specifically expressed in tumors as targets for immune intervention. This review will focus on the process from in silico analysis of expression databases and screening of potential candidate genes by bioinformatics to the in vitro and in vivo analysis to determine the immunogenicity of candidate tumor antigens. Basic biological principles of 'reverse immunology' as well as technical advantages and difficulties will be addressed.

Algorithms↗

PubMed2001

The molecular basis of inhibition and toxicity of modified cytosine analogues targetting HIV-1 reverse transcriptase.

Among the AIDS drugs approved by the US Food and Drug Administration (FDA) for clinical use, two are modified cytosine analogues, zalcitabine (ddC) and lamivudine [(-)3TC]. (-)3TC is the only analogue containing an unnatural L(-)nucleoside configuration. Similar to other dideoxynucleosides, these analogues are metabolically activated to the triphosphate that is incorporated into DNA by HIV-1 reverse transcriptase (RT), resulting in DNA chain termination and ultimately cessation of viral replication. The natural d(+)3TC isomer also acts in a similar manner to inhibit HIV-1 RT. In cell culture (-)3TC is less toxic than its d(+)isomer (+)3TC, containing the natural nucleoside configuration, and both are considerably less toxic than ddC. The mechanistic basis for the stereochemical selectivity and differential toxicity of the isomeric 3TC and ddC compounds is not completely understood, although a number of factors may clearly come into play. We have previously investigated the mechanistic basis for the differential stereoselective inhibition and toxicity of these three cytosine analogues by comparing the effects of ddCTP (+)3TC-TP and (-)3TC-TP on the HIV-1 RT, as well as a recombinant form of the human mitochondrial DNA polymerase (Polgamma), the holoenzyme polymerase responsible for mitochondrial DNA replication. In this paper, we discuss the molecular mechanism for the stereochemical selectivity and differential toxicity.

Cytosine↗

PubMed2001

Characterization of HLA-A3-restricted cytotoxic T lymphocytes reactive against the widely expressed tumor antigen telomerase.

PURPOSE: We have reported previously that the telomerase catalytic subunit, human telomerase reverse transcriptase (hTERT), is a widely expressed tumor-associated antigen recognized by CTLs. A nine-amino acid peptide derived from hTERT binds strongly to HLA-A2 antigen and elicits CTL responses against a broad panel of hTERT+ tumors (but not hTERT+ hematopoietic progenitor cells). The applicability of hTERT as a potential target for anticancer immunotherapy would be widened by the identification of epitopes restricted to other common HLA alleles, such as HLA-A3 antigen. EXPERIMENTAL DESIGN: Using a method of epitope deduction, HLA-A3-restricted peptide epitopes were screened from hTERT and tested for immunogenicity in a human in vitro T-cell system. RESULTS: The hTERT peptide K973 was used to generate specific CD8+ CTLs from HLA-A3+ cancer patients and healthy individuals. These CTLs lysed hTERT+ tumors from multiple histologies in an MHC-restricted fashion, suggesting that the epitope is naturally processed and presented by tumors. In contrast, highly enriched HLA-A3+ CD34+ peripheral blood progenitor cells or activated T cells were not lysed. CONCLUSION: Given the expression of HLA-A2 and HLA-A3 antigen in the general population, these findings extend the potential applicability of hTERT as a therapeutic target to >60% of all cancer patients. The characterization of hTERT as a polyepitope, polyallelic tumor-associated antigen may provide an approach for circumventing therapy-induced resistance potentially mediated by antigenic- and allelic-loss tumor escape mutants.

Amino Acid Sequence↗

PubMed2000

Insights into the HER-2 receptor tyrosine kinase mechanism and substrate specificity using a transient kinetic analysis.

The HER-2/erbB-2/c-neu proto-oncogene encodes for an EGF receptor-like protein which has been implicated in the pathogenesis of several human malignancies. Although much has been learned about the physiological significance of this receptor tyrosine kinase, its catalytic mechanism remains poorly understood. We have expressed, purified, and characterized two recombinant proteins corresponding to a full-length (HCD) and truncated (HKD) construct of the HER-2 intracellular tyrosine kinase domain and have identified an optimal substrate (GGMEDIYFEFMGGKKK; HER2Peptide) through screening of a degenerate peptide library. We have conducted a transient kinetic analysis of the HER-2 proteins (HCD and HKD) to illuminate mechanistic details of the HER-2 pathway. In particular, stopped-flow fluorescence studies with mant (N-methylanthraniloyl)-nucleotide derivatives provided direct measurements of the association and dissociation rate constants for these nucleotide interactions with the HER-2 recombinant proteins, thereby enabling the determination of nucleotide K(d) values. Moreover, the actual step of chemical catalysis was isolated using rapid chemical quench techniques and shown to occur approximately 3-fold faster than the steady-state rate which corresponds to product release. Evidence is also provided that suggests a conformational change that is partially rate-limiting at least in HCD. Furthermore, the role that the phosphorylation state of the protein may play on catalysis was examined. Studies carried out with pre-phosphorylated recombinant HER-2 proteins suggest that while autophosphorylation is not a prerequisite for enzymatic activity, this protein modification actually directly affects the catalytic mechanism by enhancing the rate of ADP release and that of the rate-limiting step. While a pre-steady-state kinetic analysis has been carried out on the catalytic subunit of cAMP-dependent serine/threonine kinase, to our knowledge, this study represents the first reported transient kinetic investigation of a receptor tyrosine kinase. This work serves as a basis for comparison of these two important protein kinase families and in this report we highlight these similarities and differences.

Catalysis↗

PubMed2000

Energetics of S-adenosylmethionine synthetase catalysis.

S-adenosylmethionine synthetase (ATP:L-methionine S-adenosyltransferase) catalyzes the only known route of biosynthesis of the primary biological alkylating agent. The internal thermodynamics of the Escherichia coli S-adenosylmethionine (AdoMet) synthetase catalyzed formation of AdoMet, pyrophosphate (PP(i)), and phosphate (P(i)) from ATP, methionine, and water have been determined by a combination of pre-steady-state kinetics, solvent isotope incorporation, and equilibrium binding measurements in conjunction with computer modeling. These studies provided the rate constants for substrate binding, the two chemical interconversion steps [AdoMet formation and subsequent tripolyphosphate (PPP(i)) hydrolysis], and product release. The data demonstrate the presence of a kinetically significant isomerization of the E.AdoMet.PP(i).P(i) complex before product release. The free energy profile for the enzyme-catalyzed reaction under physiological conditions has been constructed using these experimental values and in vivo concentrations of substrates and products. The free energy profile reveals that the AdoMet formation reaction, which has an equilibrium constant of 10(4), does not have well-balanced transition state and ground state energies. In contrast, the subsequent PPP(i) hydrolytic reaction is energetically better balanced. The thermodynamic profile indicates the use of binding energies for catalysis of AdoMet formation and the necessity for subsequent PPP(i) hydrolysis to allow enzyme turnover. Crystallographic studies have shown that a mobile protein loop gates access to the active site. The present kinetic studies indicate that this loop movement is rapid with respect to k(cat) and with respect to substrate binding at physiological concentrations. The uniformly slow binding rates of 10(4)-10(5) M(-)(1) s(-)(1) for ligands with different structures suggest that loop movement may be an intrinsic property of the protein rather than being ligand induced.

Adenosine Triphosphate↗

PubMed2000

Mechanism of inhibition of the human immunodeficiency virus type 1 reverse transcriptase by d4TTP: an equivalent incorporation efficiency relative to the natural substrate dTTP.

Among the clinically used nucleoside analogue inhibitors that target human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), there is little detailed mechanistic information on the interactions of 2',3'-didehydro-2', 3'-dideoxythymidine-5'-triphosphate (d4TTP) with the enzyme. primer-template complex and how these interactions compare with those of the natural substrate, dTTP. Using a pre-steady-state kinetic analysis, we found that d4TTP was incorporated by HIV-1 RT just as efficiently as dTTP during both DNA- and RNA-dependent DNA synthesis. To our knowledge, these results represent the first observation of a 3'-modified nucleoside triphosphate analogue that has an incorporation efficiency comparable to that observed for the natural substrate during DNA synthesis by HIV-1 RT. This information provides a mechanistic basis for understanding the inhibition of HIV-1 RT by d4TTP as well as insight into the clinically observed lack of d4T resistance mutations in HIV-1 RT isolated from AIDS patients.

Anti-HIV Agents↗

PubMed2000

An analysis of the catalytic cycle of HIV-1 reverse transcriptase: opportunities for chemotherapeutic intervention based on enzyme inhibition.

This review describes each of the steps in the HIV-1 reverse transcriptase catalytic cycle and evaluates each of these steps as a potential point of inhibition of the enzyme and consequently viral replication. To date, two classes of approved drugs act on the reverse transcriptase. They are: (1) the nucleoside reverse transcriptase inhibitors which either directly inhibit the enzyme or serve as alternative substrates for catalysis (resulting in chain termination) and (2) the non-nucleoside reverse transcriptase inhibitors which bind to an allosteric site and adversely affect the function of the enzyme by slowing the rate of chemical catalysis. In order to provide the best possible analysis of the potential of each of the steps in the catalytic cycle as a site of inhibition, the molecular forces which determine the intrinsic binding affinities and specificity of natural components of the catalytic complex will be described in as much detail as possible.

Animals↗

PubMed1999

Initiation of minus-strand DNA synthesis by human immunodeficiency virus type 1 reverse transcriptase.

The initiation of (-) strand DNA synthesis by HIV-1 reverse transcriptase was examined using a transient kinetic approach and a physiologically relevant RNA 18-mer/RNA 36-mer primer-template substrate. HIV-1 reverse transcriptase (RT) was found to bind with reasonably high affinity to the RNA/RNA substrate (K(d) = 90 nM), although the affinity for DNA/RNA and DNA/DNA substrates is higher (K(d) approximately 5 nM). A pre-steady-state burst of deoxynucleotide incorporation (k(obsd) = 1.0 s(-)(1)) into the RNA duplex was observed followed by a slower steady-state release of the elongated primer-template product (k(ss) = 0.58 s(-)(1)). The observation of a burst provides evidence that the release of the product is most likely the rate-limiting step in the overall kinetic pathway for the enzymatic reaction during a single deoxynucleotide incorporation event. Furthermore, the release of this product was 5-fold faster than that for elongated DNA/RNA and DNA/DNA products. Single-turnover experiments showed that there is a hyperbolic dependence of the rate of deoxynucleotide incorporation on the concentration of dCTP and demonstrated that the maximum rate of dCTP incorporation (k(pol) = 1.4 s(-)(1)) is 33- and 12-fold slower than the values for DNA/RNA and DNA/DNA primer-template substrates, respectively, while the affinity of dCTP (K(d) = 780 microM) for the HIV-1 RT.RNA/RNA complex is 56- and 71-fold weaker than the affinities for HIV-1 RT.DNA/RNA and HIV-1 RT.DNA/DNA complexes, respectively. Consequently, the overall efficiency of dCTP incorporation (k(pol)/K(d)) into the RNA/RNA substrate is approximately 1800- and 800-fold less than that for DNA/RNA and DNA/DNA substrates, respectively. These findings provide evidence which suggests that the HIV-1 RT.RNA/RNA.dCTP ternary complex exists in a significantly different conformation compared to ternary complexes involving DNA/RNA and DNA/DNA substrates. A model summarizing these results is presented, and implications for the molecular mechanism of initiation of (-) strand DNA synthesis by RT are discussed.

Binding Sites↗

PubMed1999

Crystallographic studies of phosphonate-based alpha-reaction transition-state analogues complexed to tryptophan synthase.

In an effort to use a structure-based approach for the design of new herbicides, the crystal structures of complexes of tryptophan synthase with a series of phosphonate enzyme inhibitors were determined at 2.3 A or higher resolution. These inhibitors were designed to mimic the transition state formed during the alpha-reaction of the enzyme and, as expected, have affinities much greater than that of the natural substrate indole-3-glycerol phosphate or its nonhydrolyzable analogue indole propanol phosphate (IPP). These inhibitors are ortho-substituted arylthioalkylphosphonate derivatives that have an sp(3)-hybridized sulfur atom, designed to mimic the putative tetrahedral transition state at the C3 atom of the indole, and lack the C2 atom to allow for higher conformational flexibility. Overall, the inhibitors bind in a fashion similar to that of IPP. Glu-49 and Phe-212 are the two active site residues whose conformation changes upon inhibitor binding. A very short hydrogen bond between a phosphonate oxygen and the Ser-235 hydroxyl oxygen may be responsible for stabilization of the enzyme-inhibitor complexes. Implications for the mechanism of catalysis as well as directions for more potent inhibitors are discussed.

Crystallography, X-Ray↗

PubMed1999

Mechanistic studies examining the efficiency and fidelity of DNA synthesis by the 3TC-resistant mutant (184V) of HIV-1 reverse transcriptase.

A single amino acid substitution from methionine-184 to valine (M184V) of HIV-1 reverse transcriptase (RT) evokes the 1000-fold 3TC (Lamivudine) resistance by the HIV-1 virus observed in the clinic. The M184V mutant HIV-1 RT was studied to assess its catalytic efficiency during single nucleotide incorporation using a transient kinetic approach. The maximum rate of polymerization (k(pol)), binding affinity (K(d)), and incorporation efficiency (k(pol)/K(d)) were determined for incorporating dCTP and 3TC-TP by wild-type and 3TC-resistant HIV-1 RT. The 3TC-resistant HIV-1 RT showed a similar efficiency of incorporation compared with the wild-type enzyme during DNA-dependent DNA polymerization; however, the incorporation efficiency is reduced 3.5-fold during RNA-dependent polymerization. A dramatic 146- and 117-fold decrease in incorporation efficiency was observed for 3TC-MP incorporation by M184V RT for DNA- and RNA-dependent DNA polymerization, respectively, as compared with wild-type HIV-1 RT. While the k(pol) was slower and the K(d) was weaker for 3TC-TP incorporation by the M184V RT, the decrease in the efficiency of incorporation is primarily due to a substantially reduced binding affinity for the 3TC-TP to the enzyme.DNA (or RNA) complex poised for DNA elongation. The fidelity of M184V RT was also examined to evaluate mispair formation since this mutant has been suggested to exhibit a higher level of fidelity. The results of our studies indicate that there is a maximum 2.4-fold increase in fidelity for M184V RT as compared with wild-type HIV-1 RT. Both the wild-type and 3TC-resistant mutant RT showed higher fidelity using an RNA template as contrasted with the corresponding DNA template. This mechanistic information provides insight into our understanding of the molecular mechanism of 3TC-drug resistance and supports suggestions that increased RT fidelity and decreased fitness of the M184V HIV-1 virus may be factors contributing to the strong antiviral effect of AZT-3TC combination therapy.

Amino Acid Substitution↗

PubMed1999

The catalytic mechanism of EPSP synthase revisited.

Recent analysis of EPSP synthase by solid-state NMR has led to the postulation of a new enzyme reaction pathway and raised once again the question of an intermediate species covalently bound to the enzyme [Studelska, D., McDowell, L., Espe, M., Klug, C., and Schaefer, J. (1997) Biochemistry 36, 15555-15560]. Therefore, we have reexamined the mechanism of the reaction catalyzed by EPSP synthase and analyzed the reaction products formed under the conditions used in preparing samples for solid-state NMR. Single-turnover experiments were carried out using both [1-14C]- and [32P]PEP showing the formation and decay of the previously proposed tetrahedral intermediate species on a time scale comparable with the disappearance of substrate and formation of product, thus unequivocally establishing the kinetic competence. The possible presence of a covalently bound enzyme intermediate species was also investigated, using SDS-PAGE and Centricon concentration analysis of the quenched reaction samples. No covalently bound enzyme intermediates were observed during the reaction. An enzyme assay was also performed repeating the conditions used in sample preparation for the solid-state NMR studies. We show that under these conditions, total turnover of substrates to products was observed within 45 s at -30 degrees C prior to freezing and lyophilization. Following lyophilization, the samples were stored at -20 degrees C and analyzed over a period of 21 days. We observed the conversion of the product EPSP into the side product, a cyclic EPSP ketal, and the breakdown product, pyruvate. Thus, the new species reported by solid-state NMR can be accounted for by previously characterized reaction products and side products formed during sample preparation and upon incubation in the solid-state. Our conclusions are also supported by the solution and solid-state NMR studies recently reported [Jakeman et al. (1998) Biochemistry 37, 12012-12019]. These results once again highlight the importance of kinetic competence as a criterion to be used in defining enzyme intermediates and point to the errors in interpretation of results when the time dependence of formation of the proposed intermediates is not considered.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗