Distinct Conformation-mediated Functions of an Active Site Loop in the Catalytic Reactions of NAD-dependent D-Lactate Dehydrogenase and Formate Dehydrogenase

The three-dimensional structures of NAD-dependent d-lactate dehydrogenase (d-LDH) and formate dehydrogenase (FDH), which resemble each other, imply that the two enzymes commonly employ certain main chain atoms, which are located on corresponding loop structures in the active sites of the two enzymes, for their respective catalytic functions. These active site loops adopt different conformations in the two enzymes, a difference likely attributable to hydrogen bonds with Asn97 and Glu141, which are also located at equivalent positions in d-LDH and FDH, respectively. X-ray crystallography at 2.4-Å resolution revealed that replacement of Asn97 with Asp did not markedly change the overall protein structure but markedly perturbed the conformation of the active site loop in Lactobacillus pentosus d-LDH. The Asn97 → Asp mutant d-LDH exhibited virtually the same kcat, but about 70-fold higher KM value for pyruvate than the wild-type enzyme. For Paracoccus sp. 12-A FDH, in contrast, replacement of Glu141 with Gln and Asn induced only 5.5- and 4.3-fold increases in the KM value, but 110 and 590-fold decreases in the kcat values for formate, respectively. Furthermore, these mutant FDHs, particularly the Glu141 → Asn enzyme, exhibited markedly enhanced catalytic activity for glyoxylate reduction, indicating that FDH is converted to a 2-hydroxy-acid dehydrogenase on the replacement of Glu141. These results indicate that the active site loops play different roles in the catalytic reactions of d-LDH and FDH, stabilization of substrate binding and promotion of hydrogen transfer, respectively, and that Asn97 and Glu141, which stabilize suitable loop conformations, are essential elements for proper loop functioning. The three-dimensional structures of NAD-dependent d-lactate dehydrogenase (d-LDH) and formate dehydrogenase (FDH), which resemble each other, imply that the two enzymes commonly employ certain main chain atoms, which are located on corresponding loop structures in the active sites of the two enzymes, for their respective catalytic functions. These active site loops adopt different conformations in the two enzymes, a difference likely attributable to hydrogen bonds with Asn97 and Glu141, which are also located at equivalent positions in d-LDH and FDH, respectively. X-ray crystallography at 2.4-Å resolution revealed that replacement of Asn97 with Asp did not markedly change the overall protein structure but markedly perturbed the conformation of the active site loop in Lactobacillus pentosus d-LDH. The Asn97 → Asp mutant d-LDH exhibited virtually the same kcat, but about 70-fold higher KM value for pyruvate than the wild-type enzyme. For Paracoccus sp. 12-A FDH, in contrast, replacement of Glu141 with Gln and Asn induced only 5.5- and 4.3-fold increases in the KM value, but 110 and 590-fold decreases in the kcat values for formate, respectively. Furthermore, these mutant FDHs, particularly the Glu141 → Asn enzyme, exhibited markedly enhanced catalytic activity for glyoxylate reduction, indicating that FDH is converted to a 2-hydroxy-acid dehydrogenase on the replacement of Glu141. These results indicate that the active site loops play different roles in the catalytic reactions of d-LDH and FDH, stabilization of substrate binding and promotion of hydrogen transfer, respectively, and that Asn97 and Glu141, which stabilize suitable loop conformations, are essential elements for proper loop functioning. NAD-dependent d- and l-lactate dehydrogenases (d-LDH 1The abbreviations used are: d-LDH, d-lactate dehydrogenase; d-HicDH, d-hydroxyisocaproate dehydrogenase; FDH, formate dehydrogenase; l-LDH, l-lactate dehydrogenase; MES, 2-(N-morpholino)ethanesulfonic acid. and l-LDH, EC 1.1.1.28 and EC 1.1.1.27, respectively) are evolutionally unrelated enzymes (1Taguchi H. Ohta T. J. Biol. Chem. 1991; 266: 12588-12594Abstract Full Text PDF PubMed Google Scholar, 2Bernard N. Ferain T. Garmyn D. Hols P. Delcour J. FEBS Lett. 1991; 290: 61-64Crossref PubMed Scopus (77) Google Scholar, 3Kochhar S. Hunziker P.E. Leong-Morgenthaler P. Hottinger H. J. Biol. Chem. 1992; 267: 8499-8513Abstract Full Text PDF PubMed Google Scholar) but catalyze essentially the same reaction, reduction of pyruvate into lactate concomitantly with the oxidization of NADH into NAD+, with only the chirality of the lactic acid products differing (4Holbrook J.J. Liljas A. Steindel S.J. Rossmann M.G. Boyer P.D. 3rd Ed. The Enzymes. 11. Academic Press, New York1975: 191-292Google Scholar). The two enzymes are both equipped with an imidazole-acid pair, His296– Glu264 (5Kochhar S. Chuard N. Hottinger H. J. Biol. Chem. 1992; 267: 20298-20301Abstract Full Text PDF PubMed Google Scholar, 6Taguchi H. Ohta T. J. Biol. Chem. 1993; 268: 18030-18034Abstract Full Text PDF PubMed Google Scholar, 7Taguchi H. Ohta T. J. Biochem. (Tokyo). 1994; 115: 930-936Crossref PubMed Scopus (29) Google Scholar, 8Taguchi H. Ohta T. Matsuzawa H. J. Biochem. (Tokyo). 1997; 122: 802-809Crossref PubMed Scopus (21) Google Scholar) and His195–Asp168 (4Holbrook J.J. Liljas A. Steindel S.J. Rossmann M.G. Boyer P.D. 3rd Ed. The Enzymes. 11. Academic Press, New York1975: 191-292Google Scholar), respectively (the numbering of the amino acid residues of d- and l-LDHs is according to that of Lactobacillus pentosus d-LDH (1Taguchi H. Ohta T. J. Biol. Chem. 1991; 266: 12588-12594Abstract Full Text PDF PubMed Google Scholar) and vertebrate l-LDHs (N-system) (9Eventoff W. Rossmann M.G. Taylor S.S. Torff H-J. Meyer H. Keil W. Kiltz H.-H. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 2677-2681Crossref PubMed Scopus (267) Google Scholar), respectively), as an acid/base catalyst that transfers H+ between the substrate carbonyl oxygen and the solvent. In the case of l-LDH, which is one of the best studied enzymes with respect to structure-function relationships (4Holbrook J.J. Liljas A. Steindel S.J. Rossmann M.G. Boyer P.D. 3rd Ed. The Enzymes. 11. Academic Press, New York1975: 191-292Google Scholar, 10Clarke A.R. Atkinson T. Holbrook J.J. Trends Biochem. Sci. 1989; 14 (145–148): 101-105Abstract Full Text PDF PubMed Scopus (99) Google Scholar), Arg171 and Arg109 in the substrate-binding site promote suitable substrate binding for catalysis (11Hart K.W. Clarke A.R. Wigley D.B. Waldman A.D.B. Chia W.N. Barstow D.A. Atkinson T. Jones J.B. Holbrook J.J. Biochim. Biophys. Acta. 1987; 914: 294-298Crossref PubMed Scopus (72) Google Scholar) and polarization of the bound substrate molecule (12Clarke A.R. Wigley D.B. Chia W.N. Barstow D.A. Atkinson T. Holbrook J.J. Nature. 1986; 324: 699-702Crossref PubMed Scopus (160) Google Scholar), respectively (Fig. 1A). On the other hand, the substrate-binding site of d-LDH contains only one Arg residue, Arg235, which possibly fulfills the roles of both Arg109 and Arg171 (7Taguchi H. Ohta T. J. Biochem. (Tokyo). 1994; 115: 930-936Crossref PubMed Scopus (29) Google Scholar, 8Taguchi H. Ohta T. Matsuzawa H. J. Biochem. (Tokyo). 1997; 122: 802-809Crossref PubMed Scopus (21) Google Scholar, 13Stoll V.S. Kimber M.S. Pai E.F. Structure (Lond.). 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). The three-dimensional structure that the of as Arg171 the main chain of and on a loop structure in the active which as hydrogen to the substrate V.S. Kimber M.S. Pai E.F. Structure (Lond.). 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, U. D. J. Biol. 1997; 267: PubMed Scopus Google Scholar) (Fig. d-LDH is a of the dehydrogenase with T. P. J. Biol. 1994; PubMed Scopus Google Scholar), J. Biol. Chem. 1986; Full Text PDF PubMed Google Scholar, Biochem. Biophys. 1989; PubMed Scopus Google Scholar, Biol. Scopus Google Scholar), and dehydrogenases U. D. J. Biol. 1997; 267: PubMed Scopus Google Scholar, H. J. H. J. 1989; PubMed Scopus Google Scholar) and protein S. P. 1991; PubMed Scopus Google Scholar). In d-LDH NAD-dependent formate dehydrogenase EC Biochem. Biophys. 1993; PubMed Scopus Google Scholar, V.S. J. Biol. 1994; PubMed Scopus Google Scholar, V.S. Biochem. J. 1994; PubMed Scopus Google Scholar) and dehydrogenase H. Biol. PubMed Scopus Google Scholar), than to these the 2-hydroxy-acid FDH on a formate, and to H+ between the and the FDH the residues and (the amino acid numbering is according to that for sp. FDH V.S. Biochem. J. 1994; PubMed Scopus Google at the positions corresponding to and Arg235, respectively V.S. Biochem. J. 1994; PubMed Scopus Google Scholar) (Fig. with their d-LDH these residues play different roles in with the of the acid substrate that a different in the active site V.S. J. Biol. 1994; PubMed Scopus Google Scholar, V.S. Biochem. J. 1994; PubMed Scopus Google Scholar, V.S. FEBS Lett. 1996; PubMed Scopus Google Scholar, Biochem. J. PubMed Google Scholar) (Fig. The three-dimensional structure of FDH V.S. J. Biol. 1994; PubMed Scopus Google Scholar) that the active site also contains a the main chain of the that to of d-LDH. In the case of FDH, carbonyl oxygen the bound substrate molecule (Fig. that the loop is in the promotion of polarization of the bound than substrate binding V.S. Biochem. J. 1994; PubMed Scopus Google Scholar). is that the and of Asn97 and Glu141, which are also located at corresponding positions in d-LDH and FDH, hydrogen bonds with the carbonyl and of the and main on the active site respectively V.S. Kimber M.S. Pai E.F. Structure (Lond.). 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, V.S. J. Biol. 1994; PubMed Scopus Google Scholar) (Fig. and that the of the hydrogen the at in the conformation of the active site loop in and the of the to the substrate in In a mutant pentosus d-LDH T. T. S. H. Biochem. PubMed Scopus Google Scholar), in which Asn97 with and also two mutant Paracoccus sp. 12-A and in which Glu141 with Asn and in to the roles of Asn97 and Glu141 and to the roles of the active site loops in the of the two the used for in The replacement of Asn97 with Asp in pentosus d-LDH with a in according to Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), an Paracoccus sp. 12-A FDH and the with The the chain S. A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: PubMed Scopus Google Scholar) with a to that only the of used for to to to in a H. Ohta T. J. Biol. Chem. 1993; 268: 18030-18034Abstract Full Text PDF PubMed Google Scholar) and T. T. S. H. Biochem. PubMed Scopus Google Scholar) in for the corresponding and of the enzymes essentially according to the H. Ohta T. J. Biol. Chem. 1993; 268: 18030-18034Abstract Full Text PDF PubMed Google Scholar, Nature. PubMed Scopus Google Scholar). The of the according to Nature. PubMed Scopus Google Scholar). and for at in NADH and of The activity of formate at in and of The activity of glyoxylate at in NADH and of as the of of substrate of The of NADH according to and Scopus Google Scholar) and that of acid to used for the of on the pyruvate reduction and formate the mutant d-LDH and the wild-type and mutant FDHs, respectively. at of and for and FDHs, respectively, as the amino acid and of N. Ferain T. Garmyn D. Hols P. Delcour J. FEBS Lett. 1991; 290: 61-64Crossref PubMed Scopus (77) Google Scholar) and Paracoccus sp. 12-A FDH T. T. S. H. Biochem. PubMed Scopus Google Scholar). of protein for of the d-LDH with a and of the at a of the used for the wild-type V.S. Kimber M.S. Pai E.F. Structure (Lond.). 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, V.S. Kimber M.S. U. H. Pai E.F. Lett. Scholar), the a which of and as and a protein which of the a to for X-ray at with a at the of the and the Rossmann M.G. J. 1997; Scopus Google Scholar, Biol. PubMed Scopus Google Scholar). The protein to of with of a with two The structure the protein for the pentosus d-LDH V.S. Kimber M.S. Pai E.F. Structure (Lond.). 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) and to in Biol. PubMed Scopus Google Scholar, in Scholar) the with each catalytic and NADH binding as a with of the the and used to used in the with used to each the to and in J. Biol. PubMed Scopus Google Scholar). residues of each into (Fig. the to an to an in the (1Taguchi H. Ohta T. J. Biol. Chem. 1991; 266: 12588-12594Abstract Full Text PDF PubMed Google Scholar). The contains and an of and an of and bonds are are are in a NADH to of d-LDH and NADH essentially according to a H. S. T. H. J. PubMed Scopus Google Scholar). The binding of NADH to the as the change in NADH essentially according to the used for l-LDHs Holbrook J.J. Biochem. J. PubMed Scopus Google Scholar), with and of and respectively, a the of NADH in the and of the enzymes at in The values for the enzymes with NADH according to the for l-LDHs H. S. T. H. J. PubMed Scopus Google Scholar, Holbrook J.J. Biochem. J. PubMed Scopus Google Scholar) with of Asn97 on the of pentosus is a with each two a catalytic acid positions and and a binding V.S. Kimber M.S. Pai E.F. Structure (Lond.). 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) (Fig. Asn97 is located on the loop which the two and and are located on the loop the active site loop in the catalytic of the enzyme. The hydrogen between Asn97 and and the conformation of the active site loop are in dehydrogenases (Fig. In the Lactobacillus the of a these dehydrogenases to U. D. J. Biol. 1997; 267: PubMed Scopus Google Scholar), and and in enzyme, respectively) hydrogen bonds with the of substrate (Fig. The for the protein structure and are in The structure of the exhibited in with that of the wild-type enzyme, but the catalytic of the the as with of the wild-type (Fig. difference is attributable to the differing which in the for the wild-type at in for the than the the in the and of the wild-type and enzymes for both indicating and for the in the is that the positions and which are located on in with the active site particularly and the of the hydrogen between and the and loops of the in the (Fig. and The chain of in which markedly conformations between the two is that the active site loops of the two in their conformations not only in the wild-type protein structure but also each other, particularly in the of the loop the for two of the wild-type are virtually (Fig. These indicate that the Asn97 to Asp replacement the active site loop to the is a is likely that stabilize the two active site loops of the in the different conformations in the In the case of are particularly and values at the between and (Fig. and the wild-type and in the of the and main (Fig. The hydrogen between and is and the chain of is in the in the (Fig. conformation of the active site loop in the to on the of which employ In the case of on the other hand, are and values at the between and of and and the the hydrogen between the main chain and the which is in the (Fig. The structures of Lactobacillus d-LDH A. S. Hottinger H. V.S. J. Biol. PubMed Scopus Google Scholar) and U. D. J. Biol. 1997; 267: PubMed Scopus Google Scholar) as a and an and respectively. In the structure of the enzyme, is that the of in is the main chain to the of Furthermore, in the of (Fig. in a hydrogen with the substrate with the In of the enzyme, to the main chain in a of that the wild-type the hydrogen (Fig. The of binding in a of the hydrogen to the substrate binding in the conformation of the active site The two of the also both each other and the wild-type protein in the hydrogen the active site In the wild-type enzyme, the chain of hydrogen bonds with the and carbonyl of the and main respectively. In contrast, these two hydrogen bonds are in both the two of the in which the main chain carbonyl oxygen of a hydrogen with the main chain These the conformation at positions and which also exhibited and values (Fig. In the case of the enzyme, the carbonyl oxygen of in a hydrogen with the main chain of and in and respectively, these hydrogen bonds are in the wild-type enzyme. These hydrogen to stabilize the two active site loops of the in their conformations in the The Asn97 → Asp to in of the structure of the active site loop conformation that is an of dehydrogenase The in as as the of different conformations between the two indicate that the loop conformation to in of the conformation in the wild-type to the the of residues and in the to on as the of the likely the conformation of is to on the of the structure the on the of Asn97 to Asp on the NADH of pentosus Asn97 to Asp replacement the NADH binding d-LDH, as which in the is in with the of but also to the hydrogen between and an that with the hydrogen between and NADH in the in the case of the wild-type H. S. T. H. J. PubMed Scopus Google Scholar), the of NADH markedly NADH bound to the (Fig. The for the wild-type and enzymes of and for respectively, indicating that the Asn97 to Asp replacement not binding but increases the of d-LDH for The exhibited a than the wild-type enzyme, that the Asn97 to Asp replacement the the of NADH in the enzyme. of the Asn97 to Asp on the of pentosus the of the wild-type and for and In the of the reduction of pyruvate (the substrate for the the Asn97 to Asp mutant a of reduction in the catalytic but only a change in the kcat with the → Gln and → both the kcat and values for the same substrate are (11Hart K.W. Clarke A.R. Wigley D.B. Waldman A.D.B. Chia W.N. Barstow D.A. Atkinson T. Jones J.B. Holbrook J.J. Biochim. Biophys. Acta. 1987; 914: 294-298Crossref PubMed Scopus (72) Google Scholar). The of NADH kcat and for the the enzyme. value of the is in with the value of for the wild-type (11Hart K.W. Clarke A.R. Wigley D.B. Waldman A.D.B. Chia W.N. Barstow D.A. Atkinson T. Jones J.B. Holbrook J.J. Biochim. Biophys. Acta. 1987; 914: 294-298Crossref PubMed Scopus (72) Google Scholar), but than and for the mutant enzymes, respectively (11Hart K.W. Clarke A.R. Wigley D.B. Waldman A.D.B. Chia W.N. Barstow D.A. Atkinson T. Jones J.B. Holbrook J.J. Biochim. Biophys. Acta. 1987; 914: 294-298Crossref PubMed Scopus (72) Google Scholar), that the Asn97 → Asn the is not in to catalyze the in the for for pentosus in a The in the and for the Asn to Asp replacement of the mutant of the wild-type and kcat of the mutant of the wild-type and about and respectively. These results indicate that Asn97 the active site loop are in the stabilization of the in both the and a to the Arg171 in (11Hart K.W. Clarke A.R. Wigley D.B. Waldman A.D.B. Chia W.N. Barstow D.A. Atkinson T. Jones J.B. Holbrook J.J. Biochim. Biophys. Acta. 1987; 914: 294-298Crossref PubMed Scopus (72) Google Scholar). The of for the mutant than the for the Arg171 → mutant in (11Hart K.W. Clarke A.R. Wigley D.B. Waldman A.D.B. Chia W.N. Barstow D.A. Atkinson T. Jones J.B. Holbrook J.J. Biochim. Biophys. Acta. 1987; 914: 294-298Crossref PubMed Scopus (72) Google Scholar). likely that the of in a with the of the substrate than the are of in d-LDH A.R. J. P. P. Nature. Scopus Google Scholar). In the case of d-LDH, the of the of in the substrate (7Taguchi H. Ohta T. J. Biochem. (Tokyo). 1994; 115: 930-936Crossref PubMed Scopus (29) Google Scholar, 8Taguchi H. Ohta T. Matsuzawa H. J. Biochem. (Tokyo). 1997; 122: 802-809Crossref PubMed Scopus (21) Google Scholar, 13Stoll V.S. Kimber M.S. Pai E.F. Structure (Lond.). 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) likely the of the active site loop in substrate the Arg171 in In with the case for the kcat values for markedly the Asn97 replacement the wild-type exhibited kcat values for pyruvate and the the exhibited kcat values for these a which also in the of the Arg171 replacement in (11Hart K.W. Clarke A.R. Wigley D.B. Waldman A.D.B. Chia W.N. Barstow D.A. Atkinson T. Jones J.B. Holbrook J.J. Biochim. Biophys. Acta. 1987; 914: 294-298Crossref PubMed Scopus (72) Google Scholar) and replacement in d-LDH (7Taguchi H. Ohta T. J. Biochem. (Tokyo). 1994; 115: 930-936Crossref PubMed Scopus (29) Google Scholar), that the to an for which kcat and KM values than the values in the A.R. Structure and W. H. Scholar), as in the case of the Arg171 mutant (11Hart K.W. Clarke A.R. Wigley D.B. Waldman A.D.B. Chia W.N. Barstow D.A. Atkinson T. Jones J.B. Holbrook J.J. Biochim. Biophys. Acta. 1987; 914: 294-298Crossref PubMed Scopus (72) Google Scholar). is that the to acid in an to that in which FDH formate FDH, the the between the main chain and the substrate (Fig. d-LDH a that that the of pyruvate H. S. T. H. J. PubMed Scopus Google Scholar). binding the to the in a binding in the active site but to for a different is on The exhibited a for an pyruvate as with the wild-type On the other hand, formate also the of the of the substrate pyruvate in a and the exhibited but only formate that formate is to bound to the in the which is on the active site is as formate the that the of substrate binding an with and of of Glu141 on the of FDH on the three-dimensional structure of sp. FDH V.S. J. Biol. 1994; PubMed Scopus Google Scholar), that the carbonyl oxygen of the active site loop the substrate and the V.S. Biochem. J. 1994; PubMed Scopus Google Scholar). Paracoccus sp. 12-A FDH amino acid with sp. FDH T. T. S. H. Biochem. PubMed Scopus Google Scholar). two mutant Paracoccus FDHs, the and enzymes, which in the of the Asn97 to the of the active site loop in the catalytic of the for formate the wild-type and and mutant Paracoccus sp. 12-A The and enzymes exhibited catalytic as with the wild-type enzyme, and of respectively. For both mutant enzymes, the reduction of to decreases in the kcat values than increases in the KM values than is in to the case of the Asn97 to Asp replacement in d-LDH the in converted to the with the Glu141 to Asn and to of and and of and for formate the wild-type and mutant Paracoccus sp. 12-A in a The of formate did not the KM value but to a reduction in the kcat value for the wild-type Paracoccus that the is in the catalytic of the enzyme, as in the case of FDHs, which the of V.S. Biochem. J. 1994; PubMed Scopus Google Scholar). Furthermore, the and enzymes exhibited than the wild-type These results indicate that the in kcat the Glu141 is to of the the of Glu141, and the carbonyl oxygen a active site the at of of of Glu141 on the of FDH as to FDH not acid/base catalyst as in d-LDH, which transfers H+ between substrate and solvent. the is equipped with at the corresponding to with at the of V.S. Biochem. J. 1994; PubMed Scopus Google Scholar) (Fig. for a different V.S. Biochem. J. 1994; PubMed Scopus Google Scholar, V.S. FEBS Lett. 1996; PubMed Scopus Google Scholar, Biochem. J. PubMed Google Scholar). which the catalytic of in d-LDH, is with Gln in FDH, Glu264 is not essential for the catalytic of d-LDH (5Kochhar S. Chuard N. Hottinger H. J. Biol. Chem. 1992; 267: 20298-20301Abstract Full Text PDF PubMed Google Scholar, 8Taguchi H. Ohta T. Matsuzawa H. J. Biochem. (Tokyo). 1997; 122: 802-809Crossref PubMed Scopus (21) Google Scholar), that of FDH to as an acid/base catalyst in a to of d-LDH. FDH to equipped with of the catalytic to to as a 2-hydroxy-acid for the conformation of the active site that the replacement of Glu141 the into a 2-hydroxy-acid dehydrogenase the active site loop conformation into that that in d-LDH. the activity of the mutant as to the catalytic of the wild-type and mutant enzymes for glyoxylate The wild-type exhibited catalytic activity the of which than the for formate In contrast, the and enzymes exhibited and higher for glyoxylate than the wild-type enzyme, and and higher activity for glyoxylate reduction than for formate respectively. that the amino acid replacement of Glu141 Paracoccus FDH to a glyoxylate a 2-hydroxy-acid and that the conformation of the active site loop the between FDH and 2-hydroxy-acid for glyoxylate reduction the wild-type and mutant Paracoccus sp. 12-A and indicate the values in the glyoxylate reduction and formate and indicate the values in the glyoxylate reduction and formate respectively. in a is also that the Glu141 to Asn replacement the than the Glu141 to Gln the activity glyoxylate and formate than the active site loop and a for the glyoxylate molecule to than The active site for reduction exhibited FDH is with the that and 2-hydroxy-acid dehydrogenases are for an Asn at the corresponding (Fig. is that the activity of the is to of glyoxylate the Glu141 replacement possibly hydrogen bonds with the of a substrate d-LDH with pyruvate as as (Fig. the exhibited a than KM value and a kcat for glyoxylate as with the wild-type is not is that the of glyoxylate reduction also a which kcat and KM values than the values A.R. Structure and W. H. Scholar). is that the glyoxylate molecule in the same as bound formate, particularly in the case of the wild-type enzyme, which the active site that in the substrate glyoxylate in the active site for in case only values these values are to a The change in with the to Asn replacement to of which is in with for the Asn97 to Asp replacement in d-LDH. with the wild-type enzyme, mutant FDH enzymes catalytic activity pyruvate not the active site of FDH is for formate and is than that of d-LDH V.S. J. Biol. 1994; PubMed Scopus Google Scholar, V.S. Biochem. J. 1994; PubMed Scopus Google Scholar), glyoxylate with hydrogen on is the of the to the only that the binding site of On the other hand, the d-LDH catalytic activity with respect to formate not not on the of that the active site loop of the is in a suitable conformation to formate (Fig. In d-LDH a and binding site for formate than FDH and other essential catalytic that in FDH formate as and V.S. Biochem. J. 1994; PubMed Scopus Google Scholar). The of d-LDH to FDH not to only the induced a amino acid of Asn97 in and and Glu141 in are the Lactobacillus enzymes N. Ferain T. Garmyn D. Hols P. Holbrook J.J. Delcour J. J. Biochem. 1994; PubMed Scopus Google Scholar), revealed that Asn97 is not only in but also dehydrogenases (Fig. that these enzymes employ corresponding active site loops for roles in their catalytic the also that Asn is not the only amino acid that the of is with Arg and in the of N. J. 1997; Scopus Google Scholar) and Biochim. Biophys. Acta. PubMed Scopus Google Scholar), respectively. Arg and than the to hydrogen bonds with the carbonyl of the main of these enzymes is as to their to functioning. The of Asn97 is not in the of as the and of and D. and enzymes of their amino acid with enzymes, as as which to a different Biochem. Biophys. PubMed Scopus Google Scholar). On the other hand, Glu141 is in as and (Fig. that these enzymes commonly employ the active site loop for the same that is Glu141. the main chain of a protein are in the protein is to the roles of main of protein In the of d-LDH and FDH, which resemble each other but in catalytic their main on the corresponding active site loops a that and positions the loop to the in the corresponding The results with the roles of these active site loops that on the of V.S. Kimber M.S. Pai E.F. Structure (Lond.). 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, U. D. J. Biol. 1997; 267: PubMed Scopus Google Scholar, V.S. J. Biol. 1994; PubMed Scopus Google Scholar, V.S. Biochem. J. 1994; PubMed Scopus Google Scholar). In the case of d-LDH, the main chain of the loop to stabilize the binding of a substrate in the proper for the catalytic of d-LDH with the of the in to the the chain of Arg171 in (Fig. The of and the of Asn97 in 2-hydroxy-acid that for the active site loop is a of the catalytic in In the case of FDH, on the other hand, the carbonyl on the main of the loop the of catalysis the substrate formate binding in the binding In that these of the active site loop the conformations Asn97 in d-LDH and Glu141 in is particularly that the Glu141 → Asn Paracoccus FDH to active glyoxylate indicating that the replacement at is a in the of and 2-hydroxy-acid The in the of the active site loop to in the of the in the of d-LDH and

Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.