The Biochemical Role of Glutamine 188 in Human Galactose-1-phosphate Uridyltransferase

Abstract

The substitution of arginine for glutamine at amino acid 188 (Q188R) ablates the function of human galactose-1-phosphate uridyltransferase (GALT) and is the most common mutation causing galactosemia in the white population. GALT catalyzes two consecutive reactions. The first reaction binds UDP-glucose (UDP-Glu), displaces glucose-1-phosphate (glu-1-P), and forms the UMP-GALT intermediate. In the second reaction, galactose-1-phosphate (gal-1-P) is bound, UDP-galactose (UDP-Gal) is released, and the free enzyme is recycled. In this study, we modeled glutamine, asparagine, and a common mutation arginine at amino acid 188 on the three-dimensional model of the Escherichia coli GALT-UMP protein crystal. We found that the amide group of the glutamine side chain could provide two hydrogen bonds to the phosphoryl oxygens of UMP with lengths of 2.52 and 2.82 Å. Arginine and asparagine could provide only one hydrogen bond of 2.52 and 3.02 Å, respectively. To test this model, we purified recombinant human Gln188-, Arg188-, and Asn188-GALT and analyzed the first reaction in the absence of gal-1-P by quantitating glu-1-P released using enzyme-linked methods. Gln188-GALT displaced 80 ± 7.0 nmol glu-1-P/mg GALT/min in the first reaction. By contrast, both Arg188- and Asn188-GALT released more glu-1-P (170 ± 8.0 and 129 ± 28.4 nmol/mg GALT/min, respectively). The overall, double displacement reaction was quantitated in the presence of gal-1-P. Gln188-GALT produced 80,030 ± 5,910 nmol glu-1-P/mg GALT/min, whereas the mutant Arg188- and Asn188-GALT released only 600 ± 71.2 and 2960 ± 283.6 nmole glu-1-P/mg GALT/min, respectively. We conclude from these data that glutamine at position 188 stabilizes the UMP-GALT intermediate through hydrogen bonding and enables the double displacement of both glu-1-P and UDP-Gal. The substitution of arginine or asparagine at position 188 reduces hydrogen bonding and destabilizes UMP-GALT. The unstable UMP-GALT allows single displacement of glu-1-P with release of free GALT but impairs the subsequent binding of gal-1-P and displacement of UDP-Gal. The substitution of arginine for glutamine at amino acid 188 (Q188R) ablates the function of human galactose-1-phosphate uridyltransferase (GALT) and is the most common mutation causing galactosemia in the white population. GALT catalyzes two consecutive reactions. The first reaction binds UDP-glucose (UDP-Glu), displaces glucose-1-phosphate (glu-1-P), and forms the UMP-GALT intermediate. In the second reaction, galactose-1-phosphate (gal-1-P) is bound, UDP-galactose (UDP-Gal) is released, and the free enzyme is recycled. In this study, we modeled glutamine, asparagine, and a common mutation arginine at amino acid 188 on the three-dimensional model of the Escherichia coli GALT-UMP protein crystal. We found that the amide group of the glutamine side chain could provide two hydrogen bonds to the phosphoryl oxygens of UMP with lengths of 2.52 and 2.82 Å. Arginine and asparagine could provide only one hydrogen bond of 2.52 and 3.02 Å, respectively. To test this model, we purified recombinant human Gln188-, Arg188-, and Asn188-GALT and analyzed the first reaction in the absence of gal-1-P by quantitating glu-1-P released using enzyme-linked methods. Gln188-GALT displaced 80 ± 7.0 nmol glu-1-P/mg GALT/min in the first reaction. By contrast, both Arg188- and Asn188-GALT released more glu-1-P (170 ± 8.0 and 129 ± 28.4 nmol/mg GALT/min, respectively). The overall, double displacement reaction was quantitated in the presence of gal-1-P. Gln188-GALT produced 80,030 ± 5,910 nmol glu-1-P/mg GALT/min, whereas the mutant Arg188- and Asn188-GALT released only 600 ± 71.2 and 2960 ± 283.6 nmole glu-1-P/mg GALT/min, respectively. We conclude from these data that glutamine at position 188 stabilizes the UMP-GALT intermediate through hydrogen bonding and enables the double displacement of both glu-1-P and UDP-Gal. The substitution of arginine or asparagine at position 188 reduces hydrogen bonding and destabilizes UMP-GALT. The unstable UMP-GALT allows single displacement of glu-1-P with release of free GALT but impairs the subsequent binding of gal-1-P and displacement of UDP-Gal. The enzyme galactose-1-phosphate uridyltransferase (GALT) 1The abbreviations used are: GALT, galactose-1-phosphate uridyltransferase; UDP-Glu, UDP-glucose; gal-1-P, galactose-1-phosphate; glu-1-P, glucose-1-phosphate; UDP-Gal, UDP-galactose; PAGE, polyacrylamide gel electrophoresis. (EC 2.7.7.12) catalyzes the conversion of UDP-glucose (UDP-Glu) and galactose-1-phosphate (gal-1-P) to form glucose-1-phosphate (glu-1-P) and UDP-galactose (UDP-Gal) in the evolutionarily conserved Leloir pathway of galactose metabolism (1Leloir L.F. Arch. Biochem. Biophys. 1951; 33: 186-190Crossref PubMed Scopus (189) Google Scholar). The enzymology of GALT has been intensively studied using GALT purified from Escherichia coli (2Frey P.A. Enzymes. 1992; 20: 141-186Crossref Scopus (29) Google Scholar, 3Frey P.A. FASEB J. 1996; 10: 461-470Crossref PubMed Scopus (381) Google Scholar). Both E. coli and human GALT enzymes catalyze the conversion of UDP-Glu and gal-1-P to UDP-Gal and glu-1-Pvia a double displacement mechanism (2Frey P.A. Enzymes. 1992; 20: 141-186Crossref Scopus (29) Google Scholar, 3Frey P.A. FASEB J. 1996; 10: 461-470Crossref PubMed Scopus (381) Google Scholar, 4Markus H.B. Wu J.W. Boches F.S. Tedesco T.A. Mellman W.J. Kallen R.G. J. Biol. Chem. 1977; 252: 5363-5369Abstract Full Text PDF PubMed Google Scholar) (Fig.1). Under normal physiological conditions, UDP-Glu binds to GALT to form a GALT-UDP-Glu intermediate. Glu-1-P is subsequently released, whereas the GALT enzyme remains bound to UMP. This GALT-UMP intermediate has been isolated and crystallized (5Wong L.J. Frey P.A. J. Biol. Chem. 1974; 249: 2322-2324Abstract Full Text PDF PubMed Google Scholar, 6Wedekind J.E. Frey P.A. Rayment I. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 329-331Crossref PubMed Scopus (7) Google Scholar, 7Wedekind J.E. Frey P.A. Rayment I. Biochemistry. 1995; 34: 11049-11061Crossref PubMed Scopus (84) Google Scholar, 8Wedekind J.E. Frey P.A. Rayment I. Biochemistry. 1996; 35: 11560-11569Crossref PubMed Scopus (72) Google Scholar). Gal-1-P then reacts with the GALT-UMP complex to form UDP-Gal, freeing the GALT enzyme for continued catalysis. GALT does not use nucleoside di- or triphosphates as nucleotidyl donor substrates, making it unique among nucleotidyltransferases that utilize phosphates as acceptor groups (9Wong L.J. Frey P.A. Biochemistry. 1974; 13: 3889-3894Crossref PubMed Scopus (44) Google Scholar). The GALT proteins are evolutionarily conserved; with an overall amino acid identity between the human and E. coli GALT proteins of 46%. There is near 100% conservation of the amino acid sequence in the catalytic domain studied here (10Leslie N.D. Immerman E.B. Flach J.E. Florez M. Fridovich-Keil J.L. Elsas L.J. Genomics. 1992; 14: 474-480Crossref PubMed Scopus (121) Google Scholar,11Cornwell T.L. Adhya S.L. Reznikoff W.S. Frey P.A. Nucleic Acids Res. 1987; 15: 8116Crossref PubMed Scopus (13) Google Scholar). The human GALT gene encodes 379 amino acids, contains 11 exons, and spans 4 kilobase pairs of genomic DNA (10Leslie N.D. Immerman E.B. Flach J.E. Florez M. Fridovich-Keil J.L. Elsas L.J. Genomics. 1992; 14: 474-480Crossref PubMed Scopus (121) Google Scholar). Exon VI is the most conserved domain, is composed of 19 amino acids, and is involved in these catalytic reactions (10Leslie N.D. Immerman E.B. Flach J.E. Florez M. Fridovich-Keil J.L. Elsas L.J. Genomics. 1992; 14: 474-480Crossref PubMed Scopus (121) Google Scholar). To date, 131 sequence changes have been identified in GALT genes from patients with galactosemia (12Reichardt J.K.V. Packman S. Woo S.L.C. Am. J. Hum. Genet. 1991; 49: 860-867PubMed Google Scholar, 13Segal S. Berry G.T. Scriver D. Beaudet A. Sly W. Valle D. The Metabolic Basis of Inherited Disease. McGraw-Hill, New York1995: 967-1000Google Scholar, 14Lai K. Elsas L.J. Genet. Med. 1998; 1: 40-48Abstract Full Text PDF PubMed Scopus (100) Google Scholar, 15.Lai, K., Langley, S. D., Guerrero, N. V. & Elsas, L. J. (1998), http://www.cc.emory.edu/PEDIATRICS/medgen/research/galt.htm.Google Scholar). The most common mutation is a substitution of arginine for glutamine at amino acid 188 (Q188R). This mutation is caused by an A to G transition, changing CAG to CGG in codon 188 of the highly conserved exon VI (10Leslie N.D. Immerman E.B. Flach J.E. Florez M. Fridovich-Keil J.L. Elsas L.J. Genomics. 1992; 14: 474-480Crossref PubMed Scopus (121) Google Scholar, 12Reichardt J.K.V. Packman S. Woo S.L.C. Am. J. Hum. Genet. 1991; 49: 860-867PubMed Google Scholar). This Q188R mutation has a frequency of 70% among white newborns with galactosemia, and homozygosity for the Q188R mutation has been associated with poor prognosis of the disorder (16Elsas L.J. Langley S.D. Paulk E.M. Hjelm L.H. Dembure P.P. Eur. J. Pediatr. 1995; 154: 21-27Crossref PubMed Scopus (50) Google Scholar). The precise pathobiology of this mutation remains unknown. The Gln188 is highly conserved and adjacent to three other conserved amino acids, histidine-proline-histidine at amino acids 184–186, which provide a binding domain and source for the initial nucleophilic attack in theE. coli GALT (17Field T.L. Reznikoff W.S. Frey P.A. Biochemistry. 1989; 28: 2094-2099Crossref PubMed Scopus (48) Google Scholar). A few human GALT mutations, including the Q188R mutation, have been characterized by various in vitro expression systems (12Reichardt J.K.V. Packman S. Woo S.L.C. Am. J. Hum. Genet. 1991; 49: 860-867PubMed Google Scholar,19Fridovich-Keil J.L. Langley S.D. Mazur L.A. Lennon J.C. Dembure P.P. Elsas L.J. Am. J. Hum. Genet. 1995; 56: 640-646PubMed Google Scholar, 20Fridovich-Keil J.L. Quimby B.B. Wells L. Mazur L.A. Elsevier J.P. Biochem. Mol. Med. 1996; 56: 121-130Crossref Scopus (31) Google Scholar, 21Wells L. Fridovich-Keil J.L. J. Inherit. Metab. Dis. 1996; 20: 633-642Crossref Scopus (25) Google Scholar, 36Fridovich-Keil J.L. Jinks-Robertson S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 398-402Crossref PubMed Scopus (63) Google Scholar). These studies described some kinetic parameters (apparentK M or V max) of purified mutant proteins but did not explore the normal and mutant mechanisms involved in position 188 at the In this study, we used recombinant human GALT and kinetic to the and mechanisms by which glutamine 188 to the overall reaction and the and human GALT The was used to and model human GALT at the of of E. J.E. Frey P.A. Rayment I. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 329-331Crossref PubMed Scopus (7) Google Scholar, 7Wedekind J.E. Frey P.A. Rayment I. Biochemistry. 1995; 34: 11049-11061Crossref PubMed Scopus (84) Google Scholar, 8Wedekind J.E. Frey P.A. Rayment I. Biochemistry. 1996; 35: 11560-11569Crossref PubMed Scopus (72) Google Scholar). the of this study, the of the E. coli at the was We the by the J. A. and Rayment from the for of at the of with from The E. coli used in this expression was from The of was by to a for human GALT expression and in the The human Gln188-GALT was J.E. J.K.V. Elsas L.J. Mol. Biol. Med. Google Scholar) and was by chain reaction with and The chain reaction was with enzymes and and the expression which of expression of the Gln188-GALT in E. was to the E. which 100% The recombinant was To that chain reaction, we the Gln188-GALT in both and using for the To the GALT we used and of used in this in a We the Q188R and the Gln188-GALT sequence in by the chain by and the The from glutamine to asparagine changes in two in the codon 188 This was first by of the Gln188-GALT in to form using and was then by the of the mutant GALT in to form using and the only the mutant Both the and mutant E. by the Scholar). The presence of the or mutation in the mutant was by and DNA of Both the at codon To expression of recombinant human GALT in E. coli we in of The of the was of The was to at A 600 was at a of to GALT and the was for an then by at for The expression a of in at the amino of the GALT A was used to recombinant GALT from using of the E. and New Eur. J. Biochem. PubMed Scopus Google Scholar). of with at 4 for with binding of proteins with The Gln188-, Arg188-, or Asn188-GALT proteins then with and using and in a of by with GALT proteins identified by a GALT using a described K. Langley S.D. Dembure P.P. Hjelm N. Elsas L.J. Hum. Scopus (31) Google Scholar). for the overall, double displacement reaction at in of UDP-Glu, gal-1-P, R.G. Scopus Google Scholar). The of was quantitated by at The between in and glucose-1-phosphate released was quantitated using the of of In this study, the was the was and the of for at was We in the in the To the single displacement reaction gal-1-P was with and enzymes from The 1989; PubMed Scopus Google Scholar) composed and by was used to the for the single displacement reaction. This is on the of and Biochem. J. PubMed Scopus (29) Google which is a of and for of J. Chem. Scopus Google Scholar). To an for the of the conserved amino acid Gln188 in human GALT, we modeled E. at amino acid There are more amino acids at the amino of human GALT protein as with the E. coli GALT (10Leslie N.D. Immerman E.B. Flach J.E. Florez M. Fridovich-Keil J.L. Elsas L.J. Genomics. 1992; 14: 474-480Crossref PubMed Scopus (121) Google Scholar, T.L. Adhya S.L. Reznikoff W.S. Frey P.A. Nucleic Acids Res. 1987; 15: 8116Crossref PubMed Scopus (13) Google Scholar). human Gln188 is to amino acid of E. coli GALT We found that the amide group of the glutamine side chain two hydrogen bonds to the phosphoryl oxygens of UMP with lengths of 2.52 and arginine was for glutamine at position 188 of the human GALT the model identified only one hydrogen bond of 2.52 from the amide side chain of arginine one could that arginine substitution normal of the hydrogen bonds with it has a that the GALT To for the of this we asparagine for glutamine at position is with to glutamine, side chain is one In we modeled the asparagine 188 mutation, only one hydrogen bond of 3.02 was glutamine stabilizes and the GALT-UMP by the two hydrogen we asparagine substitution to as the mutation glutamine 188 stabilizes the GALT-UMP we that asparagine or arginine substitution for glutamine with gal-1-P binding and the subsequent release of UDP-Gal in the second displacement reaction but not the release of glucose-1-phosphate in the first reaction. To these we and analyzed the two displacement reactions in vitro using purified Gln188-, Arg188-, and Asn188-GALT proteins produced in the GALT of from the expression gal-1-P not To and the first displacement reaction we the reaction of gal-1-P. gal-1-P is in the reaction, the of the first displacement reaction with gal-1-P and the second displacement reaction of human GALT in the E. coli GALT-UMP crystal. was used to model human Q188R mutation in the highly coli UMP-GALT protein crystal. The UMP in the GALT was to which is the of of the human GALT In the of the UMP was and the oxygens The side chain of glutamine which was in two hydrogen bonds of lengths 2.52 and 2.82 with the phosphoryl oxygens of the intermediate. In the amino acid glutamine was with arginine and side chain was The arginine side chain could only form one hydrogen bond of 2.52 with the phosphoryl of the intermediate. In the amino acid glutamine was with asparagine, and side chain was single displacement in the absence of gal-1-P, the for the first displacement reaction have that only of glucose-1-phosphate could from of The GALT enzyme at the of GALT-UMP intermediate. free GALT could released from the GALT-UMP it could catalyze of the first displacement reaction. a more of glucose-1-phosphate from of of the and respectively. released was quantitated by an enzyme by which of was produced by of glucose-1-phosphate We of coli for GALT by the overall double displacement reaction. Gln188-GALT an overall reaction of ± nmol glu-1-P By contrast, with Arg188- or Asn188-GALT the overall reaction for was ± or ± nmol glu-1-P The between the from the mutant Arg188- and Asn188-GALT with These to the GALT in from normal and patients are for the Q188R mutation (10Leslie N.D. Immerman E.B. Flach J.E. Florez M. Fridovich-Keil J.L. Elsas L.J. Genomics. 1992; 14: 474-480Crossref PubMed Scopus (121) Google Scholar, 12Reichardt J.K.V. Packman S. Woo S.L.C. Am. J. Hum. Genet. 1991; 49: 860-867PubMed Google Scholar). These that the at the amino of the Gln188-GALT did not the catalytic which is in with an that the amino Gln188-GALT was J.P. Fridovich-Keil J.L. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google of GALT from of GALT in glucose-1-phosphate glucose-1-phosphate in a of Gln188-, Arg188-, and Asn188-GALT in first displacement and overall acid GALT glu-1-P/mg glu-1-P/mg glu-1-P/mg ± ± ± 5,910 ± ± ± 71.2 ± ± 28.4 ± 283.6 in a we purified the recombinant human Gln188-, and proteins by E. and New Scholar, Eur. J. Biochem. PubMed Scopus Google Scholar) In 4 the three to the Gln188-GALT with of and protein are in has the of the form of GALT, and the has the of These not in the three which with from with GALT To the identity of the purified we an gel to a and using an GALT K. Langley S.D. Dembure P.P. Hjelm N. Elsas L.J. Hum. Scopus (31) Google Scholar) protein that of the two protein on with GALT K. Langley S.D. Dembure P.P. Hjelm N. Elsas L.J. Hum. Scopus (31) Google Scholar). the GALT was in with GALT protein isolated from human that recombinant human Gln188-GALT protein at two with human GALT from human K. Langley S.D. Dembure P.P. Hjelm N. Elsas L.J. Hum. Scopus (31) Google Scholar). In subsequent kinetic we that both and forms of GALT are Arch. Biochem. Biophys. PubMed Scopus Google Scholar, S. M. K. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The Arg188- or Asn188-GALT purified using the and protein by with not GALT proteins for to catalyze the overall double displacement reactions in the presence of 600 and gal-1-P. We found that the of the Gln188-GALT was ± 5,910 nmol glu-1-P/mg GALT/min By contrast, the of mutant Arg188- and Asn188-GALT 600 ± 71.2 and 2960 ± 283.6 nmol glu-1-P/mg GALT/min, arginine substitution a more asparagine substitution on GALT reaction. We a to the first displacement reaction from the overall double displacement reactions through gal-1-P in the reaction. By the reactions of gal-1-P, we that of GALT enzyme only catalyze the release of the gal-1-P and form the GALT-UMP intermediate. gal-1-P was for the second displacement reaction, the overall reaction the GALT enzyme for this only one of glucose-1-phosphate displaced by of glucose-1-phosphate was from the reaction by enzyme of glucose-1-phosphate released and subsequently produced We then a to from a of in the reaction as by the this only of produced from of glucose-1-phosphate released and of GALT could only of we have to of of GALT enzyme to a in of the GALT-UMP complex could to free UMP and GALT and the free GALT enzyme could only the first reaction with UDP-Glu the single displacement of glucose-1-phosphate could this second to a with of GALT the in only of Gln188-GALT to the single displacement reaction. data in the of three only of purified Gln188-GALT protein was used to the first displacement reaction. There was a release of glucose-1-phosphate as with the The was of the from which with the expression GALT to the a of at to a glucose-1-phosphate produced in the reaction The of purified GALT protein to the reaction was to only of GALT some of the GALT-UMP intermediate to free UMP and GALT and a single displacement reaction glucose-1-phosphate in the absence of gal-1-P. The of release of glucose-1-phosphate in the first displacement reaction was to the of the and mutant enzymes to in a of not from E. coli with the expression GALT in of in the of the reactions we UDP-Glu as in the single displacement reaction and the reaction by we found that of from of GALT, not We then the first displacement reaction using the purified mutant Arg188- and Asn188-GALT We found that the and the displacement of glucose-1-phosphate from In the mutant GALT enzymes Gln188-GALT Gln188-GALT produced 80 ± 7.0 nmol glu-1-P/mg GALT/min, whereas the Arg188- and Asn188-GALT enzymes displaced ± 8.0 and 129 ± 28.4 nmol glu-1-P/mg GALT/min, gal-1-P was to the Gln188-GALT to the overall double displacement was a of glucose-1-phosphate released a and The release of glucose-1-phosphate in the overall reaction was 80,030 ± 5,910 nmol glu-1-P/mg GALT/min, whereas only 80 ± 7.0 nmol glu-1-P/mg GALT/min was in the absence of gal-1-P This that the double displacement reaction was of the isolated single displacement reaction. gal-1-P was to the reaction the mutant Arg188- or the was a in the overall reaction the for glutamine 188 to the UMP-GALT gal-1-P, and the second displacement reaction We analyzed the of the first single displacement reaction for both and in the absence of gal-1-P We used a 1989; PubMed Scopus Google Scholar) on the of and Biochem. J. PubMed Scopus (29) Google Scholar) and and for of this the overall of at is as free GALT not UMP to form UMP-GALT J. Frey P.A. Biochemistry. PubMed Scopus Google Scholar, Frey P.A. Rayment I. Biochemistry. PubMed Scopus Google was By the to a we does not the which is glucose-1-phosphate released was to by the overall reaction, the single displacement reaction was by or for was that of Gln188-GALT nmol glu-1-P/mg nmole glu-1-P/mg one that one for the V of to that of Gln188-GALT is that the of the conversion of the intermediate to free and free UMP the of the conversion of the intermediate to free Gln188-GALT and free UMP. by it for the intermediate to free GALT the and a to the first displacement reaction is GALT is of to and a unique a double displacement reaction (2Frey P.A. Enzymes. 1992; 20: 141-186Crossref Scopus (29) Google Scholar, 3Frey P.A. FASEB J. 1996; 10: 461-470Crossref PubMed Scopus (381) Google Scholar). that amino acids and of the E. coli GALT for the first displacement reaction, but is the for the second displacement reaction (17Field T.L. Reznikoff W.S. Frey P.A. Biochemistry. 1989; 28: 2094-2099Crossref PubMed Scopus (48) Google Scholar). a mutation, found in the GALT genes of patients an catalytic by the highly conserved amino acid glutamine at position which is to glutamine in E. coli GALT (10Leslie N.D. Immerman E.B. Flach J.E. Florez M. Fridovich-Keil J.L. Elsas L.J. Genomics. 1992; 14: 474-480Crossref PubMed Scopus (121) Google Scholar, L.J. Langley S.D. Paulk E.M. Hjelm L.H. Dembure P.P. Eur. J. Pediatr. 1995; 154: 21-27Crossref PubMed Scopus (50) Google Scholar, K. Langley S.D. Hjelm N. Dembure P.P. Elsas L.J. J. Pediatr. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, J.L. Langley S.D. Mazur L.A. Lennon J.C. Dembure P.P. Elsas L.J. Am. J. Hum. Genet. 1995; 56: 640-646PubMed Google Scholar). initial of the of the amino acid glutamine to glutamine 188 in human in the of the E. coli GALT-UMP (5Wong L.J. Frey P.A. J. Biol. Chem. 1974; 249: 2322-2324Abstract Full Text PDF PubMed Google Scholar, 6Wedekind J.E. Frey P.A. Rayment I. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 329-331Crossref PubMed Scopus (7) Google Scholar, 7Wedekind J.E. Frey P.A. Rayment I. Biochemistry. 1995; 34: 11049-11061Crossref PubMed Scopus (84) Google Scholar, 8Wedekind J.E. Frey P.A. Rayment I. Biochemistry. 1996; 35: 11560-11569Crossref PubMed Scopus (72) Google Scholar) that Gln188 in the human GALT could the GALT-UMP intermediate through two hydrogen bonds between the amide side chain of Gln188 and the phosphoryl of the UMP only one hydrogen bond could in the of arginine or asparagine we subsequently that the of the GALT-UMP intermediate is for the subsequent release of UDP-Gal in the second displacement reaction. This was we and analyzed the two displacement reactions in vitro using purified recombinant Gln188-, Arg188-, and Asn188-GALT of the first displacement reaction that the GALT-UMP intermediate could to form free GALT and UMP and UDP-Glu to glucose-1-phosphate of GALT of this first displacement reaction, whereas have been These data the studies of the E. coli GALT nucleophilic attack by E. coli GALT on the UDP-Glu and displacement of glucose-1-phosphate to form the GALT-UMP intermediate (5Wong L.J. Frey P.A. J. Biol. Chem. 1974; 249: 2322-2324Abstract Full Text PDF PubMed Google Scholar, 6Wedekind J.E. Frey P.A. Rayment I. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 329-331Crossref PubMed Scopus (7) Google Scholar, 7Wedekind J.E. Frey P.A. Rayment I. Biochemistry. 1995; 34: 11049-11061Crossref PubMed Scopus (84) Google Scholar, 8Wedekind J.E. Frey P.A. Rayment I. Biochemistry. 1996; 35: 11560-11569Crossref PubMed Scopus (72) Google Scholar). A bond was modeled between the UMP and the amino acid of the E. coli GALT in the intermediate (5Wong L.J. Frey P.A. J. Biol. Chem. 1974; 249: 2322-2324Abstract Full Text PDF PubMed Google Scholar, 6Wedekind J.E. Frey P.A. Rayment I. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 329-331Crossref PubMed Scopus (7) Google Scholar, 7Wedekind J.E. Frey P.A. Rayment I. Biochemistry. 1995; 34: 11049-11061Crossref PubMed Scopus (84) Google Scholar, 8Wedekind J.E. Frey P.A. Rayment I. Biochemistry. 1996; 35: 11560-11569Crossref PubMed Scopus (72) Google Scholar). These that was a on the (5Wong L.J. Frey P.A. J. Biol. Chem. 1974; 249: 2322-2324Abstract Full Text PDF PubMed Google Scholar, 6Wedekind J.E. Frey P.A. Rayment I. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 329-331Crossref PubMed Scopus (7) Google Scholar, 7Wedekind J.E. Frey P.A. Rayment I. Biochemistry. 1995; 34: 11049-11061Crossref PubMed Scopus (84) Google Scholar, 8Wedekind J.E. Frey P.A. Rayment I. Biochemistry. 1996; 35: 11560-11569Crossref PubMed Scopus (72) Google of between and could and was by the in the group Frey P.A. Chem. 1993; Scopus Google Scholar). we gal-1-P, it was to this glucose-1-phosphate was by the enzyme-linked reactions. the bond by the group was to in the of free GALT and free UMP. To this we found that the mutant was a V the glucose-1-phosphate release in the first displacement reaction was the that forms a GALT-UMP intermediate and with Gln188-GALT was used for the first displacement reaction using the purified and mutant GALT that the mechanism of caused by the Q188R and did not binding of UDP-Glu or displacement of glucose-1-phosphate from GALT-UDP-Glu (Fig.1). These are in with the studies of the E. coli GALT Frey P.A. Rayment I. Biochemistry. PubMed Scopus Google Scholar) and kinetic of the and Gln188-GALT of for the first displacement reaction In the E. it was that was not involved in the initial binding of UDP-Glu Frey P.A. Rayment I. Biochemistry. PubMed Scopus Google Scholar). kinetic studies of the human GALT that both purified proteins with UDP-Glu, and a both enzymes the M of by and kinetic of the first displacement reaction, hydrogen bonding was a for the By contrast, the overall reaction was analyzed through release of the substitution of arginine or asparagine for glutamine at amino acid 188 GALT that to the overall double displacement reaction, a GALT-UMP intermediate is to gal-1-P and release UDP-Gal. This is by two hydrogen bonds to the phosphoryl of UMP from the amide side chain of glutamine by the single hydrogen bonds from arginine or asparagine We are to D. Langley for in of the We A. Frey and Rayment for of for the of the E. coli crystal. We are to and for on and