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G P Miller

Publications and source records attributed to G P Miller.

At least 19 recordsLinked to original sources
PubMed2001

Oxidation of phenethylamine derivatives by cytochrome P450 2D6: the issue of substrate protonation in binding and catalysis.

Cytochrome P450 (P450) 2D6 oxidizes a wide variety of drugs typically at a distance of 5-7 A from a basic nitrogen on the substrate. To investigate the determinants of P450 2D6 catalysis, we analyzed the binding and oxidation of phenethylamine substrates. P450 2D6 discriminated between the various phenethylamines, as evidenced by binding and steady-state results. Whereas the spectral binding affinity for 3-methoxyphenethylamine and 4-methoxyphenethylamine was similar, the affinity for 4-hydroxyphenethylamine was 12-fold weaker than for 3-hydroxyphenethylamine at pH 7.4. The binding of 3,4-dihydroxyphenethylamine was equally poor. These equilibrium dissociation constants were based on the observation of both type I and type II perturbation difference spectra; the former involves displacement of the proximal H(2)O ligand, yielding an iron spin state change, and the latter requires nitrogen ligation to the heme iron. One explanation for the observed type II binding spectra is the presence of both protonated and unprotonated forms of these compounds. To address this possibility, the K(S) values for 3-methoxyphenethylamine and 4-methoxyphenethylamine were determined as a function of pH. Two apparent pK(a) values were determined, which corresponded to a P450 2D6 residue involved in binding and to a lowered pK(a) of a substrate amine group upon binding P450 2D6. The apparent pK(a) of the enzyme residue (6.6) is much higher than the expected pK(a) of Asp301, which has been hypothesized to play a role in binding. Interestingly, the apparent pK(a) for the methoxyphenethylamine derivatives decreased by as much as 2 pH units upon binding to P450 2D6. 3-Methoxyphenethylamine and 4-methoxyphenethylamine underwent sequential oxidations with O-demethylation and subsequent ring hydroxylation to form 3,4-dihydroxyphenethylamine (dopamine). At higher substrate concentrations, the second oxidation was inhibited. This result can be explained by the increasing concentration of the inhibitory unprotonated substrate. Nevertheless, the rates of methoxyphenethylamine oxidations are the highest reported for P450 2D6 substrates.

Cytochrome P-450 CYP2D6↗
PubMed2001

Binding and oxidation of alkyl 4-nitrophenyl ethers by rabbit cytochrome P450 1A2: evidence for two binding sites.

Although most cytochrome P450 (P450) reactions demonstrate saturation kinetics that fit to the standard Michaelis-Menten equation, there are important exceptions where sigmoidal or nonhyperbolic behavior is observed and have been fit instead to kinetic models involving two binding sites. To assess these models, we demonstrate the consistency of a two binding site model to interpret both steady-state kinetics and binding events. Rates of 4-nitrophenol and formaldehyde production from the O-demethylation of 1-methoxy-4-nitrobenzene by P450 1A2 isolated from rabbit liver produced biphasic plots, when plotted against substrate concentration. Experiments confirmed the absence of the further oxidation of the products. Recombinant rabbit P450 1A2 yielded the same maximal velocity and more marked biphasicity. Overall, these steady-state data fit well to kinetic models involving two binding sites. Steady-state studies of substrates with bulkier O-ethyl or O-isopropoxy groups indicated decreased affinity for the second site. Based on binding studies, the affinity of P450 1A2 for these substrates increased 200-fold with the larger alkyl groups. To analyze the single binding site model, competition studies were conducted with 1,4-phenyldiisocyanide and the alkyl 4-nitrophenyl ethers. Although the observed dissociation constants and the competing titrant demonstrated a linear dependence, the affinity for the competing titrant depended on the presence of the other titrant, which violates the single binding site model. Alternatively, we applied a two binding site model to these data to obtain dissociation constants for the binary and ternary complexes. The agreement between the dissociation constants for the heterogeneous complexes supports the appropriateness of the two binding site model. This novel finding for P450 1A2 may be more common than originally perceived for P450s.

Animals↗
PubMed2001

Catalytic antibody route to the naturally occurring epothilones: total synthesis of epothilones A-F.

Naturally occurring epothilones have been synthesized starting from enantiomerically pure aldol compounds 9-11, which were obtained by antibody catalysis. Aldolase antibody 38C2 catalyzed the resolution of (+/-)-9 by enantioselective retro-aldol reaction to afford 9 in 90% ee at 50 % conversion. Compounds 10 and 11 were obtained in more than 99% ee at 50% conversion by resolution of their racemic mixtures using newly developed aldolase antibodies 84G3, 85H6 or 93F3. Compounds 9, 10 and 11 were resolved in multigram quantities and then converted to the epothilones by metathesis processes, which were catalyzed by Grubbs' catalysts.

Anti-Bacterial Agents↗
PubMed2001

Oxidations of p-alkoxyacylanilides catalyzed by human cytochrome P450 1A2: structure-activity relationships and simulation of rate constants of individual steps in catalysis.

Human cytochrome P450 (P450) 1A2 is involved in the oxidation of many important drugs and carcinogens. The prototype substrate phenacetin is oxidized to an acetol as well as the O-dealkylation product [Yun, C.-H., Miller, G. P., and Guengerich, F. P. (2000) Biochemistry 39, 11319-11329]. In an effort to improve rates of catalysis of P450 1A2 enzymes, we considered a set of p-alkoxyacylanilide analogues of phenacetin and found that variations in the O-alkyl and N-acyl substituents altered the rates of the two oxidation reactions and the ratio of acetol/phenol products. Moving one methylene group of phenacetin from the O-alkyl group to the N-acyl moiety increased rates of both oxidations approximately 5-fold and improved the coupling efficiency (oxidation products formed/NADPH consumed) from 6% to 38%. Noncompetitive kinetic deuterium isotope effects of 2-3 were measured for all O-dealkylation reactions examined with wild-type P450 1A2 and the E225I mutant, which has 6-fold higher activity. A trend of decreasing kinetic deuterium isotope effect for E225I > wild-type > mutant D320A was observed for O-demethylation of p-methoxyacetanilide, which follows the trend for k(cat). The set of O-dealkylation and acetol formation results for wild-type P450 1A2 and the E225I mutant with several of the protiated and deuterated substrates were fit to a model developed for the basic catalytic cycle and a set of microscopic rate constants in which the only variable was the rate of product formation (substrate oxygenation, including hydrogen abstraction). In this model, k(cat) is considerably less than any of the microscopic rate constants and is affected by several individual rate constants, including the rate of formation of the oxygenating species, the rate of substrate oxidation by the oxygenating species, and the rates of generation of reduced oxygen species (H(2)O(2), H(2)O). This analysis of the effects of the individual rate constants provides a framework for consideration of other P450 reactions and rate-limiting steps.

Alanine↗
PubMed2001

Interloop contacts modulate ligand cycling during catalysis by Escherichia coli dihydrofolate reductase.

As a continuation to our studies on the importance of interloop interactions in the Escherichia coli DHFR catalytic cycle, we have investigated the role of the betaG-betaH loop in modulating the closed and occluded conformations of the Met20 loop during the DHFR catalytic cycle. Specifically, to assess the importance of the hydrogen bond formed between Ser148 in the betaG-betaH loop and the Met20 loop, Ser148 was independently substituted with aspartic acid, alanine, and lysine. Moreover, the betaG-betaH loop was deleted entirely to yield the Delta(146-148) DHFR mutant. Steady-state turnover rates for all mutants were at most 3-fold lower than the wild-type rate. Lack of an isotope effect on this rate indicated the chemistry step does not contribute to the steady-state turnover. Consistent with this finding, hydride transfer rates for the DHFR mutants were at least 10-fold greater than the observed steady-state rates. The values ranged from a 30% decrease (Ser148Ala and Ser148Lys) to a 50% increase (Ser148Asp) in rate relative to that of the wild type. Modifications of the betaG-betaH loop enhanced the affinity for the cofactor and decreased the affinity for pterin, as determined by the K(D) values of the mutant proteins. Further analysis of Ser148Ala and Delta(146-148) DHFRs indicated these effects were manifest mainly in ligand off rates, although in some cases the on rate was affected. The Ser148Asp and Delta(146-148) mutations perturbed the preferred catalytic cycle through the introduction of branching at key intermediates. Rather than following the single WT pathway which involves loss of NADP(+) and rebinding of NADPH to precede loss of the product H4F (negative cooperativity), the mutants can reenter the catalytic cycle through different pathways. These findings suggest that the role of the interloop interaction between the betaG-betaH loop and the Met20 loop is to modulate ligand off rates allowing for proper cycling through the preferred kinetic pathway.

Catalysis↗
PubMed2001

Field methodology for lateral cranial radiography of nonhuman primates.

The historical lack of field-based radiographic studies of nonhuman primates within the field of anthropology is likely due to the perceived difficulty of transporting and operating X-ray equipment. Here we present a method for taking lateral cranial radiographs of nonhuman primates in the field that is simple to employ, and that produces exposed films suitable for collection of measurement data useful for growth and development studies, as well as for investigating bone and soft-tissue pathology. Several different X-ray units, film types, and portable power sources were used, all producing suitable images of similar quality, indicating that this methodology is likely not overly sensitive to these important parameters.

Animals↗
PubMed2000

Rate-determining steps in phenacetin oxidations by human cytochrome P450 1A2 and selected mutants.

Mutants with altered activities were obtained from random libraries of human cytochrome P450 (P450) 1A2 with the putative substrate recognition sequences (SRS) mutated [Parikh, A., Josephy, P. D., and Guengerich, F. P. (1999) Biochemistry 38, 5283-5289]. Six mutants from SRS 2 (E225I, E225N, F226I, and F226Y) and 4 (D320A and V322A) regions were expressed as oligohistidine-tagged proteins, purified to homogeneity, and used to analyze kinetics of individual steps in the catalytic cycle, to determine which reaction steps have been altered. When the wild-type, E225I, E225N, F226I, F226Y, D320A, and V322A proteins were reconstituted with NADPH-P450 reductase, rates of 7-ethoxyresorufin O-deethylation and phenacetin O-deethylation were in accord with those expected from membrane preparations. Within each assay, the values of k(cat)/K(m) varied by 2-3 orders of magnitude, and in the case of E225I and E225N, these parameters were 7-8-fold higher than for the wild-type enzyme. The coupling efficiency obtained from the rates of product formation and NADPH oxidation was low (<20%) in all enzymes. No correlation was found between activities and several individual steps in the catalytic cycle examined, including substrate binding, reduction kinetics, NADPH oxidation, and H(2)O(2) formation. Quench reactions did not show a burst for either phenacetin O-deethylation or formation of the acetol, a minor product, indicating that rate-determining steps occur prior to product formation. Inter- and intramolecular kinetic deuterium isotope effects for phenacetin O-deethylation were 2-3. In the case of phenacetin acetyl hydroxylation (acetol formation), large isotope effects [(D)k(cat) or (D)(k(cat)/K(m)) > 10] were observed, providing evidence for rate-limiting C-H bond cleavage. We suggest that the very high isotope effect for acetol formation reflects rate-limiting hydrogen atom abstraction; the lower isotope effect for O-deethylation may be a consequence of a 1-electron transfer pathway resulting from the low oxidation potential of the substrate phenacetin. These pre-steady-state, steady-state, and kinetic hydrogen isotope effect studies indicate that the rate-limiting steps are relatively unchanged over an 800-fold range of catalytic activity. We hypothesize that these SRS mutations alter steps leading to the formation of the activated Michaelis complex following the introduction of the first electron.

Binding Sites↗
PubMed2000

Elucidation of distinct ligand binding sites for cytochrome P450 3A4.

Cytochrome P450 (P450) 3A4 is the most abundant human P450 enzyme and has broad selectivity for substrates. The enzyme can show marked catalytic regioselectivity and unusual patterns of homotropic and heterotropic cooperativity, for which several models have been proposed. Spectral titration studies indicated one binding site for the drug indinavir (M(r) 614), a known substrate and inhibitor. Several C-terminal aminated peptides, including the model morphiceptin (YPFP-NH(2)), bind with spectral changes indicative of Fe-NH(2) bonding. The binding of the YPFP-NH(2) N-terminal amine and the influence of C-terminal modification on binding argue that the entire molecule (M(r) 521) fits within P450 3A4. YPFP-NH(2) was not oxidized by P450 3A4 but blocked binding of the substrates testosterone and midazolam, with K(i) values similar to the spectral binding constant (K(s)) for YPFP-NH(2). YPFP-NH(2) inhibited the oxidations of several typical P450 substrates with K(i) values 10-fold greater than the K(s) for binding YPFP-NH(2) and its K(i) for inhibiting substrate binding. The n values for cooperativity of these oxidations were not altered by YPFP-NH(2). YPFP-NH(2) inhibited the oxidations of midazolam at two different positions (1'- and 4-) with 20-fold different K(i) values. The differences in the K(i) values for blocking the binding to ferric P450 3A4 and the oxidation of several substrates may be attributed to weaker binding of YPFP-NH(2) to ferrous P450 3A4 than to the ferric form. The ferrous protein can be considered a distinct form of the enzyme in binding and catalysis because many substrates (but not YPFP-NH(2)) facilitate reduction of the ferric to ferrous enzyme. Our results with these peptides are considered in the context of several proposed models. A P450 3A4 model based on these peptide studies contains at least two and probably three distinct ligand sites, with testosterone and alpha-naphthoflavone occupying distinct sites. Midazolam appears to be able to bind to P450 3A4 in two modes, one corresponding to the testosterone binding mode and one postulated to reflect binding in a third site, distinct from both testosterone and alpha-naphthoflavone. The work with indinavir and YPFP-NH(2) also argues that room should be present in P450 3A4 to bind more than one smaller ligand in the "testosterone" site, although no direct evidence for such binding exists. Although this work with peptides provides evidence for the existence of multiple ligand binding sites, the results cannot be used to indicate their juxtaposition, which may vary through the catalytic cycle.

Binding Sites↗
PubMed1998

Deletion of a highly motional residue affects formation of the Michaelis complex for Escherichia coli dihydrofolate reductase.

Analysis of the dihydrofolate reductase (DHFR) complex with folate by two-dimensional heteronuclear (1H-15N) nuclear magnetic relaxation revealed that isolated residues exhibit diverse backbone fluctuations on the nanosecond to picosecond time scale [Epstein, D. M., Benkovic, S. J., and Wright, P. E. (1995) Biochemistry 34, 11037-11048]. These dynamical features may be significant in forming the Michaelis complex. Of these residues, glycine 121 displays large-amplitude backbone motions on the nanosecond time scale. This amino acid, strictly conserved for prokaryotic DHFRs, is located at the center of the betaF-betaG loop. To investigate the catalytic importance of this residue, we report the effects of Gly121 deletion and glycine insertion into the modified betaF-betaG loop. Relative to wild type, deletion of Gly121 dramatically decreases the rate of hydride transfer 550-fold and the strength of cofactor binding 20-fold for NADPH and 7-fold for NADP+. Furthermore, DeltaG121 DHFR requires conformational changes dependent on the initial binary complex to attain the Michaelis complex poised for hydride transfer. Surprisingly, the insertion mutants displayed a significant decrease in both substrate and cofactor binding. The introduction of glycine into the modified betaF-betaG loop, however, generally eliminated conformational changes required by DeltaG121 DHFR to attain the Michaelis complex. Taken together, these results suggest that the catalytic role for the betaF-betaG loop includes formation of liganded complexes and proper orientation of substrate and cofactor. Through a transient interaction with the Met20 loop, alterations of the betaF-betaG loop can orchestrate proximal and distal effects on binding and catalysis that implicate a variety of enzyme conformations participating in the catalytic cycle.

Escherichia coli↗
PubMed1998

Strength of an interloop hydrogen bond determines the kinetic pathway in catalysis by Escherichia coli dihydrofolate reductase.

On the basis of X-ray crystallographic data, Sawaya and Kraut proposed that Met20 loop conformational changes modulate ligand specificity observed in the catalytic cycle for Escherichia coli dihydrofolate reductase (DHFR) [Sawaya, M. R., and Kraut, J. (1997) Biochemistry 36, 586-603]. Interloop hydrogen bonds stabilize either a closed Met20 loop conformation observed in substrate complexes or an occluded Met20 loop conformation observed in product complexes, respectively. To test this model, we targeted a single hydrogen bond occurring exclusively in the closed Met20 loop conformation. Specifically, Asp122 in the betaF-betaG loop was independently substituted with asparagine, serine, and alanine-amino acids with decreasing abilities to hydrogen-bond. The kinetic analyses of the Asp122 mutants enabled the construction of kinetic schemes at pH 7.0 that demonstrate two striking features. First, a significant correlation exists between decreased binding of nicotinamide adenine dinucleotide phosphate, reduced (NADPH), and decreased hydride transfer rates resulting from these mutations. In other words, the interactions of Asp122 are along the reaction coordinate leading to the transition state. Second, substitutions for Asp122 alter the catalytic pathway preferred by wild-type DHFR under saturating conditions of substrate and cofactor. Overall, the steady-state rate contains contributions from the product off rates from the DHFR.5,6, 7,8-tetrahydrofolate (H4F) and DHFR.NADPH.H4F complexes and from the rate of hydride transfer. These mutational effects support the mechanistic model whereby interloop contacts regulate an equilibrium of Met20 loop conformations that, in turn, modulate ligand affinity and turnover.

Catalysis↗
PubMed1998

Stretching exercises--flexibility in dihydrofolate reductase catalysis.

As an enzyme, dihydrofolate reductase performs two tasks: transformation of its substrate dihydrofolate or folate to tetrahydrofolate, using NADPH as a cofactor, and regeneration of the enzyme for a subsequent round of catalysis. Studies discussed in this review highlight the role of conformational flexibility in both of these enzymatic functions.

Catalysis↗
PubMed1997

Expanding the 43C9 class of catalytic antibodies using a chain-shuffling approach.

We employed a chain-shuffling technique to determine if the light chain of the catalytic antibody, 43C9, provides the best partner for the 43C9 heavy chain. Previously, we reported construction and screening of a 43C9 HC CROSS library, where the 43C9 heavy-chain gene was crossed with a library of light-chain genes in a lambda bacteriophage system. The library contained a high frequency of reconstituted antibodies recognizing the transition-state analogue. Here, we report the isolation and characterization of four of these clones. Recovered light-chain proteins share 92-96% sequence identity to the 43C9 light-chain protein. Somatic mutations of these light chains occur randomly at positions distant from the active site. Residues required for binding and catalysis were conserved. Mutations affected the topology of the binding site. Nevertheless, catalysis was not affected. Isolation of these light chains suggests the best partner for the 43C9 heavy chain is the original light chain. These clones attempt to broaden a class of 43C9-like antibodies, where the catalytic residues, His91 and Arg96, have been reproducibly selected. Similar catalytic properties between the 43C9-like antibodies suggests binding has been optimized, thus further maturation of the light chain would not lead to a better catalyst. To improve catalysis, other approaches must be considered.

Amino Acid Sequence↗
PubMed1991

Immobilized microbe bioreactors for waste water treatment.

The application of adapted microbial populations immobilized on a porous diatomaceous earth carrier to pre-treat and reduce toxic concentration of volatile organics, pesticides, petroleum aliphatics and aromatics has been demonstrated for several industrial sites. In the pre-treatment of industrial effluents and contaminated groundwaters, these bioreactors have been used to optimize and reduce the cost of conventional treatment systems, i.e. steam stripping, carbon adsorption and traditional biotreatment. Additionally, these systems have been employed as seeding devices for larger biotreatment systems. The cost effective utilization of an immobilized microbe reactor system for water supply regeneration in a microgravity environment is presented. The feasibility of using immobilized biomass reactors as an effluent treatment technology for the biotransformation and biodegradation of phenols, chlorinated halocarbons, residual oils and lubricants was evaluated. Primary biotransformation tests of two benchmark toxicants, phenol and ethylene dichloride at concentrations expected in life support effluents were conducted. Biocatalyst supports were evaluated for colonization potential, surface and structural integrity, and performance in continuous flow bioreactors. The implementation of such approaches in space will be outlined and specific areas for interfacing with other non-biological treatment approaches will be considered for advanced life support, tertiary waste water biotreatment.

Biodegradation, Environmental↗
PubMed1986

The immunology of cryptococcal disease.

Cryptococcus neoformans is a ubiquitous fungus found in the soil. Upon inhalation, a complex, incompletely understood series of host responses begins that determines whether the infection will be controlled or will progress to local or disseminated disease. Local pulmonary disease may be asymptomatic or may pursue a subacute course with mild pulmonary symptoms and systemic complaints suggestive of tumor. In the compromised host, however, symptomatic pulmonary disease is often the harbinger of systemic dissemination. Early host responses include phagocytosis by polymorphonuclear leukocytes aided by complement activation which provides opsonins. Lymphocytes are activated to produce lymphokines which may enhance macrophage phagocytosis and intracellular killing of ingested cryptococci. Other lymphocytes may function as natural killer-like cells or inhibit the growth of the fungus. Production of antibody further facilitates phagocytosis by both polymorphonuclear leukocytes (PMN) and monocytes (MC). In the presence of antibody, both PMN and MC demonstrate antibody-dependent cell-mediated cytotoxicity. The combination of humoral and cell-mediated immunity in normal hosts appears to provide excellent protection against disseminated infection as evidence by the rarity of disease in exposed individuals with positive skin tests. By contrast, the frequency of cryptococcal disease in steroid-treated individuals, allograft recipients, and AIDS victims highlight the importance of T lymphocyte dependent host defenses. In view of compelling in vitro evidence for the importance of humoral responses, the infrequency of cryptococcal disease in patients with gammopathies remains a puzzle.

Cryptococcosis↗
PubMed1985

Multiparameter analysis of human lymphocyte subpopulations using flow cytometry.

This review has emphasized several recent advances in our understanding of human lymphocyte biology made possible by mAbs that recognize specific functional subpopulations of lymphocytes, coupled with multiparameter flow-cytometric analysis. In the last 3 years, both the T4+ and T8+ subsets of T cells have been further divided according to function and appearance of specific cell surface antigens. In addition, subpopulations of NK cells with different maturation states and B cells with distinct functional abilities have been discovered. Multiparameter flow-cytometric analysis of lymphocyte subpopulations can provide clues to the pathogenetic mechanisms of immune disorders, as has been demonstrated in AIDS patients and children with JRA. The use of multiparameter flow-cytometric analysis to study human immune responses in vitro has enabled a clearer understanding of the complex lymphoid interactions and, as we believe, may play an important role in the analysis of immune-associated disease processes.

Acquired Immunodeficiency Syndrome↗