Investigations on the kinetic properties of estrone glucuronyltransferase from pig kidney

Rao, Marie Luise; Rao, Govind S.; Breuer, H.

doi:10.1016/0005-2744(76)90060-7

Cited by 22 publications

(8 citation statements)

References 10 publications

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“…The buffer chamber was filled with only 600 l of blank buffer and 50 mM phosphate buffer (pH 7.4), without the test compound and the binding macromolecule. The filled up RED device was covered with parafilm and incubated at 100 rpm at 37°C for 6 to 8 h. The test compounds concentration range in the sample chamber was 5 to 750, 5 to 500, Beef and guinea pig liver microsomes p-Nitrophenol Rapid equilibrium random-order ternary complex Vessey and Zakim, 1972 Rat liver microsomes Morphine Compulsory-order ternary complex (UDPGA first, aglycone second) Sanchez and Tephly, 1975 Pig kidney microsomes Estrone Ternary-complex mechanism, iso-TheorellChance (aglycone first, UDPGA second) Rao et al, 1976 Purified UGT from rat liver Chenodeoxycholic acid and testosterone Ternary-complex mechanism Matern et al, 1982 Rat intestinal microsomes 1-Naphthol Compulsory-order ternary complex (aglycone first, UDPGA second)…”

Section: Methodsmentioning

confidence: 99%

“…To understand the overall enzyme kinetic mechanism, we have also investigated the reverse reaction, an approach that was rarely taken in previous UGT studies, particularly because recombinant UGTs became available and ensure that the analysis is conducted on a single enzyme rather than a mixture of different UGTs (Vessey and Zakim, 1972;Rao et al, 1976;Matern et al, 1991). Hence, the bisubstrate enzyme kinetics, in the presence of BSA, was also studied for the reverse reaction, namely the formation of 4-MU and UDPGA from 4-MUG and UDP (Fig.…”

Section: Bisubstrate Kinetics Of 4-mu Glucuronidation By Ugt1a9mentioning

confidence: 99%

“…Alongside the broad agreement on the formation of ternary complex, however, there is disagreement among the previous studies on whether the two substrates, the aglycone substrate and the UDPGA, bind in a random order or in a compulsory order (Table 1). The observed discrepancies in the order of substrate binding may have arisen from the use of liver microsomal preparations that contain multiple UGT enzymes (Potrepka and Spratt, 1972;Vessey and Zakim, 1972;Sanchez and Tephly, 1975;Rao et al, 1976;Koster and Noordhoek, 1983) or of partially purified UGT enzymes (Matern et al, 1982(Matern et al, , 1991Falany et al, 1987;Yin et al, 1994) that may have been inactivated by detergents during the purification process (Kurkela et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

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UDP-Glucuronic Acid Binds First and the Aglycone Substrate Binds Second to Form a Ternary Complex in UGT1A9-Catalyzed Reactions, in Both the Presence and Absence of Bovine Serum Albumin

2012

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ABSTRACT:The presence of bovine serum albumin (BSA) largely modulates the enzyme kinetics parameters of the human UDP-glucuronosyltransferase (UGT) 1A9, increasing both the apparent aglycone substrate affinity of the enzyme and its limiting reaction velocity (Drug Metab Dispos 39:2117-2129, 2011). For a better understanding of the BSA effects and an examination of whether its presence changes the catalytic mechanism, we have studied the enzyme kinetics of 4-methylumbelliferone glucuronidation by UGT1A9 in the presence and absence of 0.1% BSA, using bisubstrate enzyme kinetic experiments, in both the forward and reverse directions, as well as product and dead-end inhibition. The combined results strongly suggest that the reaction mechanism of UGT1A9, and presumably other human UGTs as well, involves the formation of a compulsoryorder ternary-complex, with UDP-␣-D-glucuronic acid (UDPGA) as the first binding substrate. Based on the enzyme kinetic parameters measured for the forward and reverse reactions, the equilibrium constant of the overall reaction was calculated (Keq ‫؍‬ 574) and the relative magnitudes of the reaction rate constants were elucidated. The inclusion of BSA in the bisubstrate kinetic experiments quantitatively changed the apparent enzyme kinetic parameters, presumably by removing internal inhibitors that bind to the binary enzyme-UDPGA (E-UDPGA) complex, as well as to the ternary E-UDPGA-aglycone complex. Nevertheless, the underlying compulsory-order ternary-complex mechanism with UDPGA binding first is the same in both the absence and presence of BSA. The results offer a novel understanding of UGT enzyme kinetic mechanism and BSA effects.

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Section: Methodsmentioning

confidence: 99%

Section: Bisubstrate Kinetics Of 4-mu Glucuronidation By Ugt1a9mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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UDP-Glucuronic Acid Binds First and the Aglycone Substrate Binds Second to Form a Ternary Complex in UGT1A9-Catalyzed Reactions, in Both the Presence and Absence of Bovine Serum Albumin

2012

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“…The three-dimensional struc-ture of the active site is unknown. A unanimous conclusion of all previous mechanistic studies is that the reaction involves the formation of a ternary complex (Potrepka and Spratt, 1972;Vessey and Zakim, 1972;Sanchez and Tephly, 1975;Rao et al, 1976;Koster and Noordhoek, 1983;Falany et al, 1987;Matern et al, 1991;Yin et al, 1994). Product and dead-end inhibition studies conducted to determine the order of substrate binding have given ambiguous results, however.…”

mentioning

confidence: 99%

Kinetic Characterization of the 1a Subfamily of Recombinant Human Udp-Glucuronosyltransferases

Luukkanen

Taskinen²,

Kurkela³

et al. 2005

Drug Metab Dispos

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ABSTRACT:The initial glucuronidation rates were determined for eight recombinant human UDP-glucuronosyltransferases (UGTs) of the 1A subfamily, and the bisubstrate kinetics and inhibition patterns were analyzed. At low substrate concentrations, the reactions followed general ternary complex kinetics, whereas at higher concentrations of both substrates, the reactions were mostly characterized by ternary complex kinetics with substrate inhibition. The glucuronidation of entacapone by UGT1A9 was inhibited by 1-naphthol in a competitive fashion, with respect to entacapone, and an uncompetitive fashion, with respect to UDP-glucuronic acid (UDPGA). Its inhibition by UDP, on the other hand, was noncompetitive with respect to entacapone and competitive with respect to UDPGA. These inhibition patterns are compatible with a compulsory ordered bi bi mechanism in which UDPGA is the firstbinding substrate. Despite the identical primary structure of the C-terminal halves of the UGT1A isoforms, there were marked differences in the respective K m values for UDPGA, ranging from 52 M for UGT1A6 to 1256 M for UGT1A8. Relative specificity constants were calculated for the eight UGT1A isoforms with 1-hydroxypyrene, 4-nitrophenol, scopoletin, 4-methylumbelliferone, and entacapone as aglycone substrates. The results demonstrated that seven of the UGT1A isoforms are capable of conjugating phenolic substrates with similar highest k cat values, and UGT1A4 has a lower relative turnover rate. The highest specificity constants were obtained for 1-hydroxypyrene, even with UGT1A6, which has been regarded as a specific isoform for small planar phenols. A k cat value of 1.9 s ؊1 was calculated for the glucuronidation of scopoletin by purified UGT1A9.

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“…As a result of substrate-inhibition studies, three specific kinetic mechanisms have been proposed for various UDPGT isoenzymes. These are a random-order ternary complex mechanism [12,26,27], a compulsory-order ternary complex mechanism [28] and a Theorell-Chance mechanism [28,29]. In the case of EGT, further work is necessary to allow a distinction to be made between the different forms of sequential mechanism.…”

Section: Discussionmentioning

confidence: 99%

Purification and kinetic analysis of a baculovirus ecdysteroid UDP-glucosyltransferase

EVANS¹,

O'REILLY²

1998

Biochemical Journal

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Investigations on the kinetic properties of estrone glucuronyltransferase from pig kidney

Cited by 22 publications

References 10 publications

UDP-Glucuronic Acid Binds First and the Aglycone Substrate Binds Second to Form a Ternary Complex in UGT1A9-Catalyzed Reactions, in Both the Presence and Absence of Bovine Serum Albumin

UDP-Glucuronic Acid Binds First and the Aglycone Substrate Binds Second to Form a Ternary Complex in UGT1A9-Catalyzed Reactions, in Both the Presence and Absence of Bovine Serum Albumin

Kinetic Characterization of the 1a Subfamily of Recombinant Human Udp-Glucuronosyltransferases

Purification and kinetic analysis of a baculovirus ecdysteroid UDP-glucosyltransferase

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