Activity and Enzyme Kinetics of Human UDP-Glucuronosyltransferases : Studies of Psilocin Glucuronidation and the Effects of Albumin on the Enzyme Kinetic Mechanism

Nenad Manevski

Research output: ThesisDoctoral ThesisCollection of Articles

Abstract

Human UDP-glucuronosyltransferases (UGTs) are important in the metabolic elimination of xenobiotics and endogenous compounds from the body. These enzymes transfer glucuronic acid moiety from the cosubstrate, UDP-glucuronic acid (UDPGA), to nucleophilic groups of small organic molecules, such as hydroxyl, carboxylic, or amino group. The conjugation of these molecules with polar glucuronic acid usually diminishes their pharmacodynamic activity, promotes aqueous solubility and enhances recognition by efflux transporters in the cells, all of which contributes to the efficient metabolic elimination and excretion of the conjugate from the body. Due to these unique properties, UGT enzymes play major roles in drug metabolism and pharmacokinetics.

The main goal of this thesis was to investigate the activity and enzyme kinetics of UGTs, as well as the in vitro assay conditions needed to accurately determine the enzyme kinetic parameters. Particular attention focused on the glucuronidation of psilocin, the enhancement of UGT activity by the inclusion of purified bovine serum albumin (BSA), and the enzyme kinetic mechanism of UGT1A9. These goals are especially important in the early phases of preclinical drug development, where in vitro assays serve to explain and predict the glucuronidation of the drug in vivo, both qualitatively and quantitatively.

As a starting point, we studied the glucuronidation of psilocin, the hallucinogenic indole alkaloid from mushrooms of the genus Psilocybe, by all the human UGTs of subfamilies 1A, 2A, and 2B. To understand the substrate selectivity of human UGTs, we also studied the glucuronidation of 4-hydroxyindole, a chemically simpler analog of psilocin which lacks the N,N-dimethylaminoethyl side chain. We successfully prevented the oxidative degradation of psilocin, a problem we encountered early in the study, by including an antioxidant (1 mM dithiothreitol) in the assays. Our results showed that psilocin is glucuronidated mainly by UGTs 1A10 and 1A9, whereas the activities of UGTs 1A8, 1A7, and 1A6 were lower. On the other hand, 4-hydroxyindole was glucuronidated mainly by UGT1A6, whereas the activities of UGTs 1A7 1A10 closely correlated with their respective rates of psilocin glucuronidation. To understand in which human tissues psilocin glucuronidation takes place, we studied the expression levels of mRNA for UGTs 1A7 1A10; this work was performed in collaboration with Dr. Michael H. Court of the Tufts University School of Medicine, Boston, Massachusetts. The combined results of the activity and expression studies indicate that although the intestinal enzyme UGT1A10 shows the highest glucuronidation clearance, UGT1A9, an enzyme abundantly expressed in both the liver and kidneys, may be the main contributor to psilocin glucuronidation in vivo.

The inclusion of purified albumin is known to significantly enhance glucuronidation rates in vitro. In subsequent studies, we focused our attention on the scope and mechanism of this activity enhancement and investigated albumin effects in a total of 11 human UGTs. Before proceeding with enzyme kinetic assays, we carefully measured the binding of substrates to BSA by either ultrafiltration or rapid equilibrium dialysis. Our results showed that the inclusion of BSA significantly enhances the in vitro glucuronidation activity of almost all the UGTs we tested, either by increasing the apparent substrate affinity (lower Km) or the reaction-limiting velocity (higher Vmax), or both. The nature of albumin effects, however, varied greatly and depended both on the UGT enzyme and the substrate employed. The highest activity increases in the presence of BSA were observed in UGTs 1A7, 1A9, 1A10, 2A1, and 2B7, whereas BSA stimulation was comparatively less pronounced in UGTs 1A1, 1A6, 1A8, 2B4, and 2B15. On the other hand, depending on the substrate used, the addition of BSA to UGTs 1A1, 1A6, and 2B17 sometimes resulted in a lack of any stimulatory effects. Moreover, the activity enhancement by BSA appears independently of the enzyme source used, since both native enzymes in human liver microsomes and recombinant enzymes expressed in Sf9 insect cells yielded similar results.

To investigate the mechanism of albumin effects, as well as to elucidate the enzyme kinetic mechanism of human UGTs, we studied bisubstrate enzyme kinetics, the product inhibition, and dead-end inhibition kinetics of UGT1A9. For this purpose, we employed 4-methylumbelliferone as the aglycone substrate and investigated both forward- and reverse-direction UGT-catalyzed reactions. The combined results of our experiments strongly suggest that UGT1A9 follows the compulsory-order ternary-complex mechanism with UDPGA binding first. The addition of BSA quantitatively changes the enzyme kinetic parameters, presumably by removing internal inhibitors that bind to binary (enzyme-UDPGA) or ternary (enzyme-UDPGA-aglycone) complexes, but the underlying compulsory-order ternary-complex mechanism remains unaffected. In addition, based on enzyme kinetic parameters measured in the forward and reverse reaction, we elucidated the thermodynamic equilibrium constant of the overall reaction (Keq = 574), as well as the relative magnitude of the individual rate constants. In summary, the results obtained deepen our current knowledge of UGT enzyme kinetics and set new guidelines for performing in vitro UGT assays. The study of psilocin and 4-hydroxyindole glucuronidation revealed that relatively small structural modifications, such as the loss of the side chain, lead to major changes in UGT substrate selectivity. And provided the substrate binding to BSA is accounted for, the addition of BSA significantly enhances the activities of almost all the UGTs tested and improves the accuracy of the measured enzyme kinetic parameters. These features are especially important for the prediction of UGT activity in vivo. Finally, our results deepen our current understanding of the UGT enzyme kinetic mechanism and conclusively show that UDPGA is the first, and the aglycone substrate is the second binding substrate to form a ternary complex in UGT1A9-catalyzed reactions.
Original languageEnglish
Place of PublicationHelsinki
Publisher
Print ISBNs978-952-10-8624-3
Electronic ISBNs978-952-10-8625-0
Publication statusPublished - 2013
MoE publication typeG5 Doctoral dissertation (article)

Fields of Science

  • 116 Chemical sciences
  • 317 Pharmacy
  • 319 Forensic science and other medical sciences
  • 1182 Biochemistry, cell and molecular biology

Cite this

@phdthesis{de96628e81bd45dfb0964aaeb3656d9d,
title = "Activity and Enzyme Kinetics of Human UDP-Glucuronosyltransferases : Studies of Psilocin Glucuronidation and the Effects of Albumin on the Enzyme Kinetic Mechanism",
abstract = "Human UDP-glucuronosyltransferases (UGTs) are important in the metabolic elimination of xenobiotics and endogenous compounds from the body. These enzymes transfer glucuronic acid moiety from the cosubstrate, UDP-glucuronic acid (UDPGA), to nucleophilic groups of small organic molecules, such as hydroxyl, carboxylic, or amino group. The conjugation of these molecules with polar glucuronic acid usually diminishes their pharmacodynamic activity, promotes aqueous solubility and enhances recognition by efflux transporters in the cells, all of which contributes to the efficient metabolic elimination and excretion of the conjugate from the body. Due to these unique properties, UGT enzymes play major roles in drug metabolism and pharmacokinetics. The main goal of this thesis was to investigate the activity and enzyme kinetics of UGTs, as well as the in vitro assay conditions needed to accurately determine the enzyme kinetic parameters. Particular attention focused on the glucuronidation of psilocin, the enhancement of UGT activity by the inclusion of purified bovine serum albumin (BSA), and the enzyme kinetic mechanism of UGT1A9. These goals are especially important in the early phases of preclinical drug development, where in vitro assays serve to explain and predict the glucuronidation of the drug in vivo, both qualitatively and quantitatively. As a starting point, we studied the glucuronidation of psilocin, the hallucinogenic indole alkaloid from mushrooms of the genus Psilocybe, by all the human UGTs of subfamilies 1A, 2A, and 2B. To understand the substrate selectivity of human UGTs, we also studied the glucuronidation of 4-hydroxyindole, a chemically simpler analog of psilocin which lacks the N,N-dimethylaminoethyl side chain. We successfully prevented the oxidative degradation of psilocin, a problem we encountered early in the study, by including an antioxidant (1 mM dithiothreitol) in the assays. Our results showed that psilocin is glucuronidated mainly by UGTs 1A10 and 1A9, whereas the activities of UGTs 1A8, 1A7, and 1A6 were lower. On the other hand, 4-hydroxyindole was glucuronidated mainly by UGT1A6, whereas the activities of UGTs 1A7 1A10 closely correlated with their respective rates of psilocin glucuronidation. To understand in which human tissues psilocin glucuronidation takes place, we studied the expression levels of mRNA for UGTs 1A7 1A10; this work was performed in collaboration with Dr. Michael H. Court of the Tufts University School of Medicine, Boston, Massachusetts. The combined results of the activity and expression studies indicate that although the intestinal enzyme UGT1A10 shows the highest glucuronidation clearance, UGT1A9, an enzyme abundantly expressed in both the liver and kidneys, may be the main contributor to psilocin glucuronidation in vivo. The inclusion of purified albumin is known to significantly enhance glucuronidation rates in vitro. In subsequent studies, we focused our attention on the scope and mechanism of this activity enhancement and investigated albumin effects in a total of 11 human UGTs. Before proceeding with enzyme kinetic assays, we carefully measured the binding of substrates to BSA by either ultrafiltration or rapid equilibrium dialysis. Our results showed that the inclusion of BSA significantly enhances the in vitro glucuronidation activity of almost all the UGTs we tested, either by increasing the apparent substrate affinity (lower Km) or the reaction-limiting velocity (higher Vmax), or both. The nature of albumin effects, however, varied greatly and depended both on the UGT enzyme and the substrate employed. The highest activity increases in the presence of BSA were observed in UGTs 1A7, 1A9, 1A10, 2A1, and 2B7, whereas BSA stimulation was comparatively less pronounced in UGTs 1A1, 1A6, 1A8, 2B4, and 2B15. On the other hand, depending on the substrate used, the addition of BSA to UGTs 1A1, 1A6, and 2B17 sometimes resulted in a lack of any stimulatory effects. Moreover, the activity enhancement by BSA appears independently of the enzyme source used, since both native enzymes in human liver microsomes and recombinant enzymes expressed in Sf9 insect cells yielded similar results. To investigate the mechanism of albumin effects, as well as to elucidate the enzyme kinetic mechanism of human UGTs, we studied bisubstrate enzyme kinetics, the product inhibition, and dead-end inhibition kinetics of UGT1A9. For this purpose, we employed 4-methylumbelliferone as the aglycone substrate and investigated both forward- and reverse-direction UGT-catalyzed reactions. The combined results of our experiments strongly suggest that UGT1A9 follows the compulsory-order ternary-complex mechanism with UDPGA binding first. The addition of BSA quantitatively changes the enzyme kinetic parameters, presumably by removing internal inhibitors that bind to binary (enzyme-UDPGA) or ternary (enzyme-UDPGA-aglycone) complexes, but the underlying compulsory-order ternary-complex mechanism remains unaffected. In addition, based on enzyme kinetic parameters measured in the forward and reverse reaction, we elucidated the thermodynamic equilibrium constant of the overall reaction (Keq = 574), as well as the relative magnitude of the individual rate constants. In summary, the results obtained deepen our current knowledge of UGT enzyme kinetics and set new guidelines for performing in vitro UGT assays. The study of psilocin and 4-hydroxyindole glucuronidation revealed that relatively small structural modifications, such as the loss of the side chain, lead to major changes in UGT substrate selectivity. And provided the substrate binding to BSA is accounted for, the addition of BSA significantly enhances the activities of almost all the UGTs tested and improves the accuracy of the measured enzyme kinetic parameters. These features are especially important for the prediction of UGT activity in vivo. Finally, our results deepen our current understanding of the UGT enzyme kinetic mechanism and conclusively show that UDPGA is the first, and the aglycone substrate is the second binding substrate to form a ternary complex in UGT1A9-catalyzed reactions.",
keywords = "116 Chemical sciences, 317 Pharmacy, 319 Forensic science and other medical sciences, 1182 Biochemistry, cell and molecular biology",
author = "Nenad Manevski",
year = "2013",
language = "English",
isbn = "978-952-10-8624-3",
series = "Dissertationes Biocentri Viikki Universitatis Helsingiensis",
publisher = "University of Helsinki",
number = "6",
address = "Finland",

}

Activity and Enzyme Kinetics of Human UDP-Glucuronosyltransferases : Studies of Psilocin Glucuronidation and the Effects of Albumin on the Enzyme Kinetic Mechanism. / Manevski, Nenad.

Helsinki : University of Helsinki, 2013. 97 p.

Research output: ThesisDoctoral ThesisCollection of Articles

TY - THES

T1 - Activity and Enzyme Kinetics of Human UDP-Glucuronosyltransferases : Studies of Psilocin Glucuronidation and the Effects of Albumin on the Enzyme Kinetic Mechanism

AU - Manevski, Nenad

PY - 2013

Y1 - 2013

N2 - Human UDP-glucuronosyltransferases (UGTs) are important in the metabolic elimination of xenobiotics and endogenous compounds from the body. These enzymes transfer glucuronic acid moiety from the cosubstrate, UDP-glucuronic acid (UDPGA), to nucleophilic groups of small organic molecules, such as hydroxyl, carboxylic, or amino group. The conjugation of these molecules with polar glucuronic acid usually diminishes their pharmacodynamic activity, promotes aqueous solubility and enhances recognition by efflux transporters in the cells, all of which contributes to the efficient metabolic elimination and excretion of the conjugate from the body. Due to these unique properties, UGT enzymes play major roles in drug metabolism and pharmacokinetics. The main goal of this thesis was to investigate the activity and enzyme kinetics of UGTs, as well as the in vitro assay conditions needed to accurately determine the enzyme kinetic parameters. Particular attention focused on the glucuronidation of psilocin, the enhancement of UGT activity by the inclusion of purified bovine serum albumin (BSA), and the enzyme kinetic mechanism of UGT1A9. These goals are especially important in the early phases of preclinical drug development, where in vitro assays serve to explain and predict the glucuronidation of the drug in vivo, both qualitatively and quantitatively. As a starting point, we studied the glucuronidation of psilocin, the hallucinogenic indole alkaloid from mushrooms of the genus Psilocybe, by all the human UGTs of subfamilies 1A, 2A, and 2B. To understand the substrate selectivity of human UGTs, we also studied the glucuronidation of 4-hydroxyindole, a chemically simpler analog of psilocin which lacks the N,N-dimethylaminoethyl side chain. We successfully prevented the oxidative degradation of psilocin, a problem we encountered early in the study, by including an antioxidant (1 mM dithiothreitol) in the assays. Our results showed that psilocin is glucuronidated mainly by UGTs 1A10 and 1A9, whereas the activities of UGTs 1A8, 1A7, and 1A6 were lower. On the other hand, 4-hydroxyindole was glucuronidated mainly by UGT1A6, whereas the activities of UGTs 1A7 1A10 closely correlated with their respective rates of psilocin glucuronidation. To understand in which human tissues psilocin glucuronidation takes place, we studied the expression levels of mRNA for UGTs 1A7 1A10; this work was performed in collaboration with Dr. Michael H. Court of the Tufts University School of Medicine, Boston, Massachusetts. The combined results of the activity and expression studies indicate that although the intestinal enzyme UGT1A10 shows the highest glucuronidation clearance, UGT1A9, an enzyme abundantly expressed in both the liver and kidneys, may be the main contributor to psilocin glucuronidation in vivo. The inclusion of purified albumin is known to significantly enhance glucuronidation rates in vitro. In subsequent studies, we focused our attention on the scope and mechanism of this activity enhancement and investigated albumin effects in a total of 11 human UGTs. Before proceeding with enzyme kinetic assays, we carefully measured the binding of substrates to BSA by either ultrafiltration or rapid equilibrium dialysis. Our results showed that the inclusion of BSA significantly enhances the in vitro glucuronidation activity of almost all the UGTs we tested, either by increasing the apparent substrate affinity (lower Km) or the reaction-limiting velocity (higher Vmax), or both. The nature of albumin effects, however, varied greatly and depended both on the UGT enzyme and the substrate employed. The highest activity increases in the presence of BSA were observed in UGTs 1A7, 1A9, 1A10, 2A1, and 2B7, whereas BSA stimulation was comparatively less pronounced in UGTs 1A1, 1A6, 1A8, 2B4, and 2B15. On the other hand, depending on the substrate used, the addition of BSA to UGTs 1A1, 1A6, and 2B17 sometimes resulted in a lack of any stimulatory effects. Moreover, the activity enhancement by BSA appears independently of the enzyme source used, since both native enzymes in human liver microsomes and recombinant enzymes expressed in Sf9 insect cells yielded similar results. To investigate the mechanism of albumin effects, as well as to elucidate the enzyme kinetic mechanism of human UGTs, we studied bisubstrate enzyme kinetics, the product inhibition, and dead-end inhibition kinetics of UGT1A9. For this purpose, we employed 4-methylumbelliferone as the aglycone substrate and investigated both forward- and reverse-direction UGT-catalyzed reactions. The combined results of our experiments strongly suggest that UGT1A9 follows the compulsory-order ternary-complex mechanism with UDPGA binding first. The addition of BSA quantitatively changes the enzyme kinetic parameters, presumably by removing internal inhibitors that bind to binary (enzyme-UDPGA) or ternary (enzyme-UDPGA-aglycone) complexes, but the underlying compulsory-order ternary-complex mechanism remains unaffected. In addition, based on enzyme kinetic parameters measured in the forward and reverse reaction, we elucidated the thermodynamic equilibrium constant of the overall reaction (Keq = 574), as well as the relative magnitude of the individual rate constants. In summary, the results obtained deepen our current knowledge of UGT enzyme kinetics and set new guidelines for performing in vitro UGT assays. The study of psilocin and 4-hydroxyindole glucuronidation revealed that relatively small structural modifications, such as the loss of the side chain, lead to major changes in UGT substrate selectivity. And provided the substrate binding to BSA is accounted for, the addition of BSA significantly enhances the activities of almost all the UGTs tested and improves the accuracy of the measured enzyme kinetic parameters. These features are especially important for the prediction of UGT activity in vivo. Finally, our results deepen our current understanding of the UGT enzyme kinetic mechanism and conclusively show that UDPGA is the first, and the aglycone substrate is the second binding substrate to form a ternary complex in UGT1A9-catalyzed reactions.

AB - Human UDP-glucuronosyltransferases (UGTs) are important in the metabolic elimination of xenobiotics and endogenous compounds from the body. These enzymes transfer glucuronic acid moiety from the cosubstrate, UDP-glucuronic acid (UDPGA), to nucleophilic groups of small organic molecules, such as hydroxyl, carboxylic, or amino group. The conjugation of these molecules with polar glucuronic acid usually diminishes their pharmacodynamic activity, promotes aqueous solubility and enhances recognition by efflux transporters in the cells, all of which contributes to the efficient metabolic elimination and excretion of the conjugate from the body. Due to these unique properties, UGT enzymes play major roles in drug metabolism and pharmacokinetics. The main goal of this thesis was to investigate the activity and enzyme kinetics of UGTs, as well as the in vitro assay conditions needed to accurately determine the enzyme kinetic parameters. Particular attention focused on the glucuronidation of psilocin, the enhancement of UGT activity by the inclusion of purified bovine serum albumin (BSA), and the enzyme kinetic mechanism of UGT1A9. These goals are especially important in the early phases of preclinical drug development, where in vitro assays serve to explain and predict the glucuronidation of the drug in vivo, both qualitatively and quantitatively. As a starting point, we studied the glucuronidation of psilocin, the hallucinogenic indole alkaloid from mushrooms of the genus Psilocybe, by all the human UGTs of subfamilies 1A, 2A, and 2B. To understand the substrate selectivity of human UGTs, we also studied the glucuronidation of 4-hydroxyindole, a chemically simpler analog of psilocin which lacks the N,N-dimethylaminoethyl side chain. We successfully prevented the oxidative degradation of psilocin, a problem we encountered early in the study, by including an antioxidant (1 mM dithiothreitol) in the assays. Our results showed that psilocin is glucuronidated mainly by UGTs 1A10 and 1A9, whereas the activities of UGTs 1A8, 1A7, and 1A6 were lower. On the other hand, 4-hydroxyindole was glucuronidated mainly by UGT1A6, whereas the activities of UGTs 1A7 1A10 closely correlated with their respective rates of psilocin glucuronidation. To understand in which human tissues psilocin glucuronidation takes place, we studied the expression levels of mRNA for UGTs 1A7 1A10; this work was performed in collaboration with Dr. Michael H. Court of the Tufts University School of Medicine, Boston, Massachusetts. The combined results of the activity and expression studies indicate that although the intestinal enzyme UGT1A10 shows the highest glucuronidation clearance, UGT1A9, an enzyme abundantly expressed in both the liver and kidneys, may be the main contributor to psilocin glucuronidation in vivo. The inclusion of purified albumin is known to significantly enhance glucuronidation rates in vitro. In subsequent studies, we focused our attention on the scope and mechanism of this activity enhancement and investigated albumin effects in a total of 11 human UGTs. Before proceeding with enzyme kinetic assays, we carefully measured the binding of substrates to BSA by either ultrafiltration or rapid equilibrium dialysis. Our results showed that the inclusion of BSA significantly enhances the in vitro glucuronidation activity of almost all the UGTs we tested, either by increasing the apparent substrate affinity (lower Km) or the reaction-limiting velocity (higher Vmax), or both. The nature of albumin effects, however, varied greatly and depended both on the UGT enzyme and the substrate employed. The highest activity increases in the presence of BSA were observed in UGTs 1A7, 1A9, 1A10, 2A1, and 2B7, whereas BSA stimulation was comparatively less pronounced in UGTs 1A1, 1A6, 1A8, 2B4, and 2B15. On the other hand, depending on the substrate used, the addition of BSA to UGTs 1A1, 1A6, and 2B17 sometimes resulted in a lack of any stimulatory effects. Moreover, the activity enhancement by BSA appears independently of the enzyme source used, since both native enzymes in human liver microsomes and recombinant enzymes expressed in Sf9 insect cells yielded similar results. To investigate the mechanism of albumin effects, as well as to elucidate the enzyme kinetic mechanism of human UGTs, we studied bisubstrate enzyme kinetics, the product inhibition, and dead-end inhibition kinetics of UGT1A9. For this purpose, we employed 4-methylumbelliferone as the aglycone substrate and investigated both forward- and reverse-direction UGT-catalyzed reactions. The combined results of our experiments strongly suggest that UGT1A9 follows the compulsory-order ternary-complex mechanism with UDPGA binding first. The addition of BSA quantitatively changes the enzyme kinetic parameters, presumably by removing internal inhibitors that bind to binary (enzyme-UDPGA) or ternary (enzyme-UDPGA-aglycone) complexes, but the underlying compulsory-order ternary-complex mechanism remains unaffected. In addition, based on enzyme kinetic parameters measured in the forward and reverse reaction, we elucidated the thermodynamic equilibrium constant of the overall reaction (Keq = 574), as well as the relative magnitude of the individual rate constants. In summary, the results obtained deepen our current knowledge of UGT enzyme kinetics and set new guidelines for performing in vitro UGT assays. The study of psilocin and 4-hydroxyindole glucuronidation revealed that relatively small structural modifications, such as the loss of the side chain, lead to major changes in UGT substrate selectivity. And provided the substrate binding to BSA is accounted for, the addition of BSA significantly enhances the activities of almost all the UGTs tested and improves the accuracy of the measured enzyme kinetic parameters. These features are especially important for the prediction of UGT activity in vivo. Finally, our results deepen our current understanding of the UGT enzyme kinetic mechanism and conclusively show that UDPGA is the first, and the aglycone substrate is the second binding substrate to form a ternary complex in UGT1A9-catalyzed reactions.

KW - 116 Chemical sciences

KW - 317 Pharmacy

KW - 319 Forensic science and other medical sciences

KW - 1182 Biochemistry, cell and molecular biology

M3 - Doctoral Thesis

SN - 978-952-10-8624-3

T3 - Dissertationes Biocentri Viikki Universitatis Helsingiensis

PB - University of Helsinki

CY - Helsinki

ER -

Manevski N. Activity and Enzyme Kinetics of Human UDP-Glucuronosyltransferases : Studies of Psilocin Glucuronidation and the Effects of Albumin on the Enzyme Kinetic Mechanism. Helsinki: University of Helsinki, 2013. 97 p. (Dissertationes Biocentri Viikki Universitatis Helsingiensis; 6).