Sequence specific interaction of Mycobacterium smegmatis topoisomerase I with duplex DNA

We have identified strong topoisomerase sites (STS) for Mycobacteruim smegmatis topoisomerase I in double-stranded DNA context using electrophoretic mobility shift assay of enzyme-DNA covalent complexes; Mg2+, an essential component for DNA relaxation activity of the enzyme, is not required for binding to DNA, The enzyme makes single-stranded nicks, with transient covalent interaction at the 5'-end of the broken DNA strand, a characteristic akin to prokaryotic topoisomerases. More importantly, the enzyme binds to duplex DNA having a preferred site with high affinity, a. property similar to the eukaryotic type I topoisomerases, The preferred cleavage site is mapped on a 65 bp duplex DNA and found to be CG/TCTT. Thus, the enzyme resembles other prokaryotic type I topoisomerases in mechanistics of the reaction, but is similar to eukaryotic enzymes in DNA recognition properties.

Nonlinear Enzyme Kinetics Can Lead to High Metabolic Flux Control Coefficients: Implications for the Evolution of Dominance

In a classic study, Kacser & Burns (1981, Genetics 97, 639-666) demonstrated that given certain plausible assumptions, the flux in a metabolic pathway was more or less indifferent to the activity of any of the enzymes in the pathway taken singly. It was inferred from this that the observed dominance of most wild-type alleles with respect to loss-of-function mutations did not require an adaptive, meaning selectionist, explanation. Cornish-Bowden (1987, J. theor. Biol. 125, 333-338) showed that the Kacser-Burns inference was not valid when substrate concentrations were large relative to the relevant Michaelis constants. We find that in a randomly constructed functional pathway, even when substrate levels are small, one can expect high values of control coefficients for metabolic flux in the presence of significant nonlinearities as exemplified by enzymes with Hill coefficients ranging from two to six, or by the existence of oscillatory loops. Under these conditions the flux can be quite sensitive to changes in enzyme activity as might be caused by inactivating one of the two alleles in a diploid. Therefore, the phenomenon of dominance cannot be a trivial ''default'' consequence of physiology but must be intimately linked to the manner in which metabolic networks have been moulded by natural selection.

Heme biosynthesis by the malarial parasite - Import of delta-aminolevulinate dehydrase from the host red cell

The mouse and human malarial parasites, Plasmodium berghei and Plasmodium falciparum, respectively, synthesize heme de novo following the standard pathway observed in animals despite the availability of large amounts of heme, derived from red cell hemoglobin, which is stored as hemozoin pigment, The enzymes, delta-aminolevulinate dehydrase (ALAD), coproporphyrinogen oxidase, and ferrochelatase are present at strikingly high levels in the P, berghei infected mouse red cell in vivo, The isolated parasite has low levels of ALAD and the data clearly indicate it to be of red cell origin. The purified enzyme preparations from the uninfected red cell and the parasite are identical in kinetic properties, subunit molecular weight, cross-reaction with antibodies to the human enzyme, and N-terminal amino acid sequence. Immunogold electron microscopy of the infected culture indicates that the enzyme is present inside the parasite and, therefore, is not a contaminant, The parasite derives functional ALAD from the host and the enzyme binds specifically to isolated parasite membrane in vitro, suggestive of the involvement of a receptor in its translocation into the parasite, While, ALAD, coproporphyrinogen oxidase, and ferrochelatase from the parasite and the uninfected red cell supernatant have identical subunit molecular weights on SDS-polyacrylamide gel electrophoresis and show immunological cross-reaction with antibodies to the human enzymes, as revealed by Western analysis, the first enzyme of the pathway, namely, delta-aminolevulinate synthase (ALAS) in the parasite, unlike that of the red cell host, does not cross-react with antibodies to the human enzyme, However, ALAS enzyme activity in the parasite is higher than that of the infected red cell supernatant. We therefore conclude that the parasite, while making its own ALAS, imports ALAD and perhaps most of the other enzymes of the pathway from the host to synthesize heme de novo, and this would enable it to segregate this heme from the heme derived from red cell hemoglobin degradation, ALAS of the parasite and the receptor(s) involved in the translocation of the host enzymes into the parasite would be unique drug targets.

The role of lysine-41 in RNase A catalysis - A quantum chemical study on the active site ligand complex

Several mechanisms have been proposed to explain the action of enzymes at the atomic level. Among them, the recent proposals involving short hydrogen bonds as a step in catalysis by Gerlt and Gassman [1] and proton transfer through low barrier hydrogen bonds (LBHBs) [2, 3] have attracted attention. There are several limitations to experimentally testing such hypotheses, Recent developments in computational methods facilitate the study of active site-ligand complexes to high levels of accuracy, Our previous studies, which involved the docking of the dinucleotide substrate UpA to the active site of RNase A [4, 5], enabled us to obtain a realistic model of the ligand-bound active site of RNase A. From these studies, based on empirical potential functions, we were able to obtain the molecular dynamics averaged coordinates of RNase A, bound to the ligand UpA. A quantum mechanical study is required to investigate the catalytic process which involves the cleavage and formation of covalent bonds. In the present study, we have investigated the strengths of some of the hydrogen bonds between the active site residues of RNase A and UpA at the ab initio quantum chemical level using the molecular dynamics averaged coordinates as the starting point. The 49 atom system and other model systems were optimized at the 3-21G level and the energies of the optimized systems were obtained at the 6-31G* level. The results clearly indicate the strengthening of hydrogen bonds between neutral residues due to the presence of charged species at appropriate positions. Such a strengthening manifests itself in the form of short hydrogen bonds and a low barrier for proton transfer. In the present study, the proton transfer between the 2'-OH of ribose (from the substrate) and the imidazole group from the H12 of RNase A is influenced by K41, which plays a crucial role in strengthening the neutral hydrogen bond, reducing the barrier for proton transfer.

Variations in the levels of aminoacylation and modified nucleotide content between total tRNAs from chloroplasts and etioplasts in cucumber cotyledons

Total tRNAs isolated from chloroplasts and etioplasts of cucumber cotyledons were compared with respect toamino acid acceptance, isoacceptor distribution and extent of modification. Aminoacylation of the tRNAs with nine different amino acids studied indicated that the relative acceptor activities of chloroplast total tRNAs for four amino acids are significantly higher than etioplast total tRNAs. Two dimensional polyacrylamide gel electrophoresis(2D-PAGE) of chloroplast total tRNAs separated at least 32 spots, while approximately 41 spots were resolved from etioplast total tRNAs. Comparison of the reversed-phase chromatography (RPC-5) profiles of chloroplast and etioplast leucyl-, lysyl-, phenylalanyl-, and valyl-tRNA species showed no qualitative differences in the elution profiles. However, leucyl-, lysyl- and valyl-tRNA species showed quantitative differences in the relative amounts of the isoaccepting species present in chloroplasts and etioplasts. The analysis of modified nucleotides of total tRNAs from the two plastid types indicated that total tRNA from etioplasts was undermodified with respect to ribothymidine, isopentenyladenosine/hydroxy-isopentenyladenosine, 1 -methylguanosine and 2-o-methylguanosine. This indicates that illumination may cause de novo synthesis of chloroplast tRNAmodifying enzymes encoded for by nuclear genes leading to the formation of highly modified tRNAs in chloroplasts. Based on these results, we speculate that the observed decrease in levels of aminoacylation, variations in the relative amounts of certain isoacceptors, and differences in the electrophoretic mobilities of some extra tRNA spots in the etioplast total tRNAs as compared to chloroplast total tRNAs could be due to some partially undermodified etioplast tRNAs. Taken together, the data suggested that the light-induced transformation of etioplasts into chloroplasts is accompanied by increases in the relative levels of some functional chloroplast tRNAs by post transcriptional nucleotide modifications.

Serum immunoglobulin G directed against porcine trypsin in the serum of cystic fibrosis children receiving porcine pancreatic enzyme supplements

Cystic fibrosis (CF) patients require pancreatic enzyme replacement therapy to correct pancreatic insufficiency. These enzymes are derived from porcine pancreas and are known to be antigenic. To determine the possible clinical consequences, a specific ELISA was developed to detect IgG antibody directed against porcine trypsin (PTAb) in the sera of CF patients. The assay was used to evaluate the occurrence of PTAb in a cross sectional study of 103 CF patients in relation to the introduction of porcine enzyme therapy, clinical status and genotype. Antibodies against porcine trypsin were detected in the sera of 63% of patients unrelated to the age of commencement or the duration of enzyme therapy. No differences were observed in the clinical status of CF patients who had developed PTAb (n = 65) and those who had no detectable PTAb (n = 38) as determined from: the current prescribed dose of porcine pancreatic enzyme capsules; Z scores for height and weight; and respiratory function tests. It is suggested that the PTAb commonly found in the sera of CF patients are of doubtful clinical significance but the prospect of PTAb contributing to immune complex disease should be examined further.

A soluble preparation from developing groundnut seeds (Arachis hypogaea) catalyzes de novo synthesis of long chain fatty acids

A 100,000 x g supernatant fraction prepared from developing groundnut seeds (30-35 days after flowering) catalyzed the synthesis of fatty acids from [l-14C]acetate at a rate of 120nmoles of acetate incorporated per hr per gram fresh weight of tissue. 90% of this incorporated label was associated with fatty acids. The major fatty acids formed were stearic- (77%) and palmitic acids (14%) with 4% of oleic acid. The fatty acid synthetase activity was stable when stored at 0-4 degrees C for at least fifteen days. It is concluded from these results that acetyl-coA carboxylase and all the enzymes of fatty acid synthetase from developing groundnut seeds are soluble.

Importance of amino-terminus in maintenance of oligomeric structure of cytosolic serine hydroxymethyltransferase

The role of the amino and carboxyl-terminal regions of cytosolic serine hydroxymethyltransferase (SHMT) in subunit assembly and catalysis was studied using six amino-terminal (lacking the first 6, 14, 30, 49, 58, and 75 residues) and two carboxyl-terminal (lacking the last 49 and 185 residues) deletion mutants. These mutants were constructed from a full length cDNA clone using restriction enzyme/PCR-based methods and overexpressed in Escherichia coli. The overexpressed proteins, des-(A1-K6)-SHMT and des-(A1- W14)-SHMT were present in the soluble fraction and they were purified to homogeneity. The deletion clones, for des-(A1–V30)-SHMT and des-(A1–L49)-SHMT were expressed at very low levels, whereas des-(A1–R58)-SHMT, des-(A1–G75)-SHMT, des-(Q435–F483)-SHMT and des-(L299-F483)-SHMT mutant proteins were not soluble and formed inclusion bodies. Des-(A1–K6)-SHMT and des-(A1–W14)-SHMT catalyzed both the tetrahydrofolate-dependent and tetrahydrofolate-independent reactions, generating characteristic spectral intermediates with glycine and tetrahydrofolate. The two mutants had similar kinetic parameters to that of the recombinant SHMT (rSHMT). However, at 55 °C, the des-(A1–W14)-SHMT lost almost all the activity within 5 min, while at the same temperature rSHMT and des-(A1–K6)-SHMT retained 85% and 70% activity, respectively. Thermal denaturation studies showed that des-(A1–W14)-SHMT had a lower apparent melting temperature (52°C) compared to rSHMT (56°C) and des-(A1–K6)-SHMT (55 °C), suggesting that N-terminal deletion had resulted in a decrease in the thermal stability of the enzyme. Further, urea induced inactivation of the enzymes revealed that 50% inactivation occurred at a lower urea concentration (1.2 ± 0.1 M) in the case of des-(A1–W14)-SHMT compared to rSHMT (1.8 ±0.1 M) and des-(A1–K6)-SHMT (1.7 ±0.1 M). The apoenzyme of des-(A1- W14)-SHMT was present predominantly in the dimer form, whereas the apoenzymes of rSHMT and des-(A1–K6)-SHMT were a mixture of tetramers (≈75% and ≈65%, respectively) and dimers. While, rSHMT and des-(A1–K6)-SHMT apoenzymes could be reconstituted upon the addition of pyridoxal-5'-phosphate to 96% and 94% enzyme activity, respectively, des-(A1–W14)-SHMT apoenzyme could be reconstituted only upto 22%. The percentage activity regained correlated with the appearance of visible CD at 425 nm and with the amount of enzyme present in the tetrameric form upon reconstitution as monitored by gel filtration. These results demonstrate that, in addition to the cofactor, the N-terminal arm plays an important role in stabilizing the tetrameric structure of SHMT.

Importance of the amino terminus in maintenance of oligomeric structure of sheep liver cytosolic serine hydroxymethyltransferase

The Role Of The Amino And Carboxyl-Terminal Regions Of Cytosolic Serine Hydroxymethyltransferase (SHMT) In Subunit Assembly And Catalysis Was Studied Using Sis Amino-Terminal (Lacking The First 6, 14, 30, 49, 58, And 75 Residues) And Two Carboxyl-Terminal (Lacking The Last 49 And 185 Residues) Deletion Mutants. These Mutants Were Constructed From A Full Length Cdna Clone Using Restriction Enzyme/PCR-Based Methods And Overexpressed In Escherichia Coli. The Overexpressed Proteins, Des-(A1-K6) SHMT And Des-(A1-W14)-SHMT Were Present In The Soluble Fraction And They Were Purified To Homogeneity. The Deletion Clones, For Des-(A1-V30)-SHMT And Des-(A1-L49)-SHMT Were Expressed At Very Low Levels, Whereas Des-(A1-R58)-SHMT, Des-/A1-G75)-SHMT, Des-(Q435-F483)-SHMT And Des-(L299-F483)-SHMT Mutant Proteins Were Not Soluble And Formed Inclusion Bodies. Des-(A1-K6)-SHMT And Des-(A1-W14)-SHMT Catalyzed Both The Tetrahydrofolate-Dependent And Tetrahydrofolate-Independent Reactions, Generating Characteristic Spectral Intermediates With Glycine And Tetrahydrofolate. The Two Mutants Had Similar Kinetic Parameters To That Of The Recombinant SHMT (Rshmt). However, At 55 Degrees C, The Des-(A1-W14)-SHMT Lost Almost All The Activity Within 5 Min, While At The Same Temperature Rshmt And Des-(A1-K6)-SHMT Retained 85% And 70% Activity, Respectively. Thermal Denaturation Studies Showed That Des-(A1-W14)-SHMT Had A Lower Apparent Melting Temperature (52 Degrees C) Compared To Rshmt (56 Degrees C) And Des-(A1-K6)-SHMT (55 Degrees C), Suggesting That N-Terminal Deletion Had Resulted In A Decrease In The Thermal Stability Of The Enzyme. Further Urea Induced Inactivation Of The Enzymes Revealed That 50% Inactivation Occurred At A Lower Urea Concentration (1.2+/-0.1 M) In The Case Of Des-(A1-W14)-SHMT Compared To Rshmt (1.8+/-0.1 M) And Des-(A1 -K6)-SHMT (1.7+/-0.1 M). The Apoenzyme Of Des-/A1-K6)-SHMT Was Present Predominantly In The Dimer Form, Whereas The Apoenzymes Of Rshmt And Des-(A1-K6)-SHMT Were A Mixture Of Tetramers (Approximate To 75% And Approximate To 65%, Respectively) And Dimers. While, Rshmt And Des-(A1-K6)-SHMT Apoenzymes Could Be Reconstituted Upon The Addition Of Pyridoxal-5'-Phosphate To 96% And 94% Enzyme Activity, Respectively Des-(A1-W14)-SHMT Apoenzyme Could Be Reconstituted Only Upto 22%. The Percentage Activity Refined Correlated With The Appearance Of Visible CD At 425 Nm And With The Amount Of Enzyme Present In The Tetrameric Form Upon Reconstitution As Monitored By Gel Filtration. These Results Demonstrate That, In Addition To The Cofactor, The N-Terminal Arm Plays An Important Role In Stabilizing The Tetrameric Structure Of SHMT.

Mycobacterium tuberculosis pantothenate kinase: possible changes in location of ligands during enzyme action

The crystal structures of complexes of Mycobacterium tuberculosis pantothenate kinase with the following ligands have been determined: (i) citrate; (ii) the nonhydrolysable ATP analogue AMPPCP and pantothenate (the initiation complex); (iii) ADP and phosphopantothenate resulting from phosphorylation of pantothenate by ATP in the crystal (the end complex); (iv) ATP and ADP, each with half occupancy, resulting from a quick soak of crystals in ATP (the intermediate complex); (v) CoA; (vi) ADP prepared by soaking and cocrystallization, which turned out to have identical structures, and (vii) ADP and pantothenate. Solution studies on CoA binding and catalytic activity have also been carried out. Unlike in the case of the homologous Escherichia coli enzyme, AMPPCP and ADP occupy different, though overlapping, locations in the respective complexes; the same is true of pantothenate in the initiation complex and phosphopantothenate in the end complex. The binding site of MtPanK is substantially preformed, while that of EcPanK exhibits considerabl plasticity. The difference in the behaviour of the E. coli and M. tuberculosis enzymes could be explained in terms of changes in local structure resulting from substitutions. It is unusual for two homologous enzymes to exhibit such striking differences in action. Therefore, the results have to be treated with caution. However, the changes in the locations of ligands exhibited by M. tuberculosis pantothenate kinase are remarkable and novel.

Purification and functional characterization of type II DNA topoisomerase from rat testis and comparison with topoisomerase II from liver

A number of studies in yeast have shown that DNA topoisomerase TI is essential for chromosome condensation and disjunction during mitosis at the metaphase/anaphase transition and meiosis I. Accordingly, kinetic and mechanistic studies have implied a role for topoisomerase rr in chromosome disjunction. As a step toward understanding the nature and role of topoisomerase II in a mammalian germline in vivo, we have purified topoisomerase II from rat testis to homogeneity and ascertained several of its catalytic activities in conjunction with that of the purified enzyme from liver. The purified enzymes appeared to be monomers under denaturing conditions; however, they differed in their relative molecular mass. Topoisomerase II from testis and liver have apparent molecular masses of 150 +/- 10 kDa and 160 +/- 10 kDa, respectively. The native molecular mass of testis topoisomerase II as assayed by immunoblot analysis of cell-foe extracts, prepared in the presence of SDS and a number of protease inhibitors, corroborated with the size of the purified enzyme. Both enzymes are able to promote decatenation and relax supercoiled DNA substrates in an ATP and Mg2+-dependent manner. However, quantitative comparison of catalytic properties of topoisomerase II from testis with that of the enzyme from liver displayed significant differences in their efficiencies. Optimal pH values for testis enzyme are 6.5 to 8.5 while they are 6 to 7.5 for the liver enzyme. Intriguingly, the relaxation activity of liver topoisomerase II was inhibited by potassium glutamate at 1 M, whereas testis enzyme required about half its concentration. These findings argue that topoisomerase II from rat testis is structurally distinct from that of its somatic form and the functional differences between the two enzymes parallels with the physiological environment that is unique to these two tissues.

Roles of forced nicotine exposure and Comt gene disruption in the development of addiction-related behavioural and neurochemical changes in mice

Activation of midbrain dopamine systems is thought to be critically involved in the addictive properties of abused substances. Drugs of abuse increase dopamine release in the nucleus accumbens and dorsal striatum, which are the target areas of mesolimbic and nigrostriatal dopamine pathways, respectively. Dopamine release in the nucleus accumbens is thought to mediate the attribution of incentive salience to rewards, and dorsal striatal dopamine release is involved in habit formation. In addition, changes in the function of prefrontal cortex (PFC), the target area of mesocortical dopamine pathway, may skew information processing and memory formation such that the addict pays an abnormal amount of attention to drug-related cues. In this study, we wanted to explore how long-term forced oral nicotine exposure or the lack of catechol-O-methyltransferase (COMT), one of the dopamine metabolizing enzymes, would affect the functioning of these pathways. We also wanted to find out how the forced nicotine exposure or the lack of COMT would affect the consumption of nicotine, alcohol, or cocaine. First, we studied the effect of forced chronic nicotine exposure on the sensitivity of dopamine D2-like autoreceptors in microdialysis and locomotor activity experiments. We found that the sensitivity of these receptors was unchanged after forced oral nicotine exposure, although an increase in the sensitivity was observed in mice treated with intermittent nicotine injections twice daily for 10 days. Thus, the effect of nicotine treatment on dopamine autoreceptor sensitivity depends on the route, frequency, and time course of drug administration. Second, we investigated whether the forced oral nicotine exposure would affect the reinforcing properties of nicotine injections. The chronic nicotine exposure did not significantly affect the development of conditioned place preference to nicotine. In the intravenous self-administration paradigm, however, the nicotine-exposed animals self-administered nicotine at a lower unit dose than the control animals, indicating that their sensitivity to the reinforcing effects of nicotine was enhanced. Next, we wanted to study whether the Comt gene knock-out animals would be a suitable model to study alcohol and cocaine consumption or addiction. Although previous work had shown male Comt knock-out mice to be less sensitive to the locomotor-activating effects of cocaine, the present study found that the lack of COMT did not affect the consumption of cocaine solutions or the development of cocaine-induced place preference. However, the present work did find that male Comt knock-out mice, but not female knock-out mice, consumed ethanol more avidly than their wild-type littermates. This finding suggests that COMT may be one of the factors, albeit not a primary one, contributing to the risk of alcoholism. Last, we explored the effect of COMT deficiency on dorsal striatal, accumbal, and prefrontal cortical dopamine metabolism under no-net-flux conditions and under levodopa load in freely-moving mice. The lack of COMT did not affect the extracellular dopamine concentrations under baseline conditions in any of the brain areas studied. In the prefrontal cortex, the dopamine levels remained high for a prolonged time after levodopa treatment in male, but not female, Comt knock-out mice. COMT deficiency induced accumulation of 3,4-dihydroxyphenylacetic acid, which increased further under levodopa load. Homovanillic acid was not detectable in Comt knock-out animals either under baseline conditions or after levodopa treatment. Taken together, the present results show that although forced chronic oral nicotine exposure affects the reinforcing properties of self-administered nicotine, it is not an addiction model itself. COMT seems to play a minor role in dopamine metabolism and in the development of addiction under baseline conditions, indicating that dopamine function in the brain is well-protected from perturbation. However, the role of COMT becomes more important when the dopaminergic system is challenged, such as by pharmacological manipulation.

Liquid Chromatography-Mass Spectrometry in Studies of Drug Metabolism and Permeability

Poor pharmacokinetics is one of the reasons for the withdrawal of drug candidates from clinical trials. There is an urgent need for investigating in vitro ADME (absorption, distribution, metabolism and excretion) properties and recognising unsuitable drug candidates as early as possible in the drug development process. Current throughput of in vitro ADME profiling is insufficient because effective new synthesis techniques, such as drug design in silico and combinatorial synthesis, have vastly increased the number of drug candidates. Assay technologies for larger sets of compounds than are currently feasible are critically needed. The first part of this work focused on the evaluation of cocktail strategy in studies of drug permeability and metabolic stability. N-in-one liquid chromatography-tandem mass spectrometry (LC/MS/MS) methods were developed and validated for the multiple component analysis of samples in cocktail experiments. Together, cocktail dosing and LC/MS/MS were found to form an effective tool for increasing throughput. First, cocktail dosing, i.e. the use of a mixture of many test compounds, was applied in permeability experiments with Caco-2 cell culture, which is a widely used in vitro model for small intestinal absorption. A cocktail of 7-10 reference compounds was successfully evaluated for standardization and routine testing of the performance of Caco-2 cell cultures. Secondly, cocktail strategy was used in metabolic stability studies of drugs with UGT isoenzymes, which are one of the most important phase II drug metabolizing enzymes. The study confirmed that the determination of intrinsic clearance (Clint) as a cocktail of seven substrates is possible. The LC/MS/MS methods that were developed were fast and reliable for the quantitative analysis of a heterogenous set of drugs from Caco-2 permeability experiments and the set of glucuronides from in vitro stability experiments. The performance of a new ionization technique, atmospheric pressure photoionization (APPI), was evaluated through comparison with electrospray ionization (ESI), where both techniques were used for the analysis of Caco-2 samples. Like ESI, also APPI proved to be a reliable technique for the analysis of Caco-2 samples and even more flexible than ESI because of the wider dynamic linear range. The second part of the experimental study focused on metabolite profiling. Different mass spectrometric instruments and commercially available software tools were investigated for profiling metabolites in urine and hepatocyte samples. All the instruments tested (triple quadrupole, quadrupole time-of-flight, ion trap) exhibited some good and some bad features in searching for and identifying of expected and non-expected metabolites. Although, current profiling software is helpful, it is still insufficient. Thus a time-consuming largely manual approach is still required for metabolite profiling from complex biological matrices.

Stereochemical and steric control of enzymatic glucuronidation : A rational approach for the design of novel Inhibitors for the human UDP-glucuronosyltransferase 2B7

The UDP-glucuronosyltransferases (UGTs) are enzymes of the phase II metabolic system. These enzymes catalyze the transfer of α-D-glucuronic acid from UDP-glucuronic acid to aglycones bearing nucleophilic groups affording exclusively their corresponding β-D-glucuronides to render lipophilic endobiotics and xenobiotics more water soluble. This detoxification pathway aids in the urinary and biliary excretion of lipophilic compounds thus preventing their accumulation to harmful levels. The aim of this study was to investigate the effect of stereochemical and steric features of substrates on the glucuronidation catalyzed by UGTs 2B7 and 2B17. Furthermore, this study relates to the design and synthesis of novel, selective inhibitors that display high affinity for the key enzyme involved in drug glucuronidation, UGT2B7. The starting point for the development of inhibitors was to assess the influence of the stereochemistry of substrates on the UGT-catalyzed glucuronidation reaction. A set of 28 enantiomerically pure alcohols was subjected to glucuronidation assays employing the human UGT isoforms 2B7 and 2B17. Both UGT enzymes displayed high stereoselectivity, favoring the glucuronidation of the (R)-enantiomers over their respective mirror-image compounds. The spatial arrangement of the hydroxy group of the substrate determined the rate of the UGT-catalyzed reaction. However, the affinity of the enantiomeric substrates to the enzymes was not significantly influenced by the spatial orientation of the nucleophilic hydroxy group. Based on these results, a rational approach for the design of inhibitors was developed by addressing the stereochemical features of substrate molecules. Further studies showed that the rate of the enzymatic glucuronidation of substrates was also highly dependent on the steric demand in vicinity of the nucleophilic hydroxy group. These findings provided a rational approach to turn high-affinity substrates into true UGT inhibitors by addressing stereochemical and steric features of substrate molecules. The tricyclic sesquiterpenols longifolol and isolongifolol were identified as high-affinity substrates which displayed high selectivity for the UGT isoform 2B7. These compounds served therefore as lead structures for the design of potent and selective inhibitors for UGT2B7. Selective and potent inhibitors were prepared by synthetically modifying the lead compounds longifolol and isolongifolol taking stereochemical and steric features into account. The best inhibitor of UGT2B7, β-phenyllongifolol, displayed an inhibition constant of 0.91 nM.

Enzymatic activity other than lipase

In this chapter, enzymes (other than lipase) which are present in cream are discussed. The effects of heat treatments on the activities of these enzymes are described. The influence of residual enzyme activiv, remaining after heating, on cream quality is also discussed.