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Identification of an arginine residue in the dual coenzyme-specific glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides that plays a key role in binding NADP+ but not NAD+.
Arch Biochem Biophys. 1996 Feb 01; 326(1):145-51.AB

Abstract

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides can utilize either NADP or NAD as coenzyme. The enzyme's three-dimensional structure has been solved (Rowland et al., 1994, Structure 2, 1073-1087) and shown to contain a conventional nucleotide binding domain. NADP+ was modeled into the structure by superimposing the beta alpha beta domain and that of coenzyme-bound 6-phosphogluconate dehydrogenase (Adams et al., 1994, Structure 2, 651-658), enabling us to identify Arg-46 as a potentially important residue for NADP+ binding. Using site-directed mutagenesis, we constructed mutant enzymes in which Arg-46 was replaced by glutamine (R46Q) and alanine (R46A) and examined their kinetic properties. The principal effects in these mutant enzymes were that the Km and Ki values for NADP+ increased by 2 to 3 orders of magnitude over those of the wild-type enzyme. No other kinetic constant was altered more than 6.5-fold. Changing this single amino acid leads to mutant glucose-6-phosphate dehydrogenases with coenzyme specificities that favor NAD+, whereas the wild-type enzyme prefers NADP+ as coenzyme. These results confirm that Arg-46 plays a key role in NADP+ binding by contributing a positively charged planar residue that interacts primarily with the 2'-adenosine phosphate. The Arg residue corresponding to Arg-46 in L. mesenteroides glucose-6-phosphate dehydrogenase is conserved in all glucose-6-phosphate dehydrogenases and, presumably, plays the same role in all these enzymes.

Authors+Show Affiliations

Department of Biology, Syracuse University, New York 13244-1220, USA. hrlevy@mailbox.syr.eduNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

8579362

Citation

Levy, H R., et al. "Identification of an Arginine Residue in the Dual Coenzyme-specific Glucose-6-phosphate Dehydrogenase From Leuconostoc Mesenteroides That Plays a Key Role in Binding NADP+ but Not NAD+." Archives of Biochemistry and Biophysics, vol. 326, no. 1, 1996, pp. 145-51.
Levy HR, Vought VE, Yin X, et al. Identification of an arginine residue in the dual coenzyme-specific glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides that plays a key role in binding NADP+ but not NAD+. Arch Biochem Biophys. 1996;326(1):145-51.
Levy, H. R., Vought, V. E., Yin, X., & Adams, M. J. (1996). Identification of an arginine residue in the dual coenzyme-specific glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides that plays a key role in binding NADP+ but not NAD+. Archives of Biochemistry and Biophysics, 326(1), 145-51.
Levy HR, et al. Identification of an Arginine Residue in the Dual Coenzyme-specific Glucose-6-phosphate Dehydrogenase From Leuconostoc Mesenteroides That Plays a Key Role in Binding NADP+ but Not NAD+. Arch Biochem Biophys. 1996 Feb 1;326(1):145-51. PubMed PMID: 8579362.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Identification of an arginine residue in the dual coenzyme-specific glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides that plays a key role in binding NADP+ but not NAD+. AU - Levy,H R, AU - Vought,V E, AU - Yin,X, AU - Adams,M J, PY - 1996/2/1/pubmed PY - 1996/2/1/medline PY - 1996/2/1/entrez SP - 145 EP - 51 JF - Archives of biochemistry and biophysics JO - Arch. Biochem. Biophys. VL - 326 IS - 1 N2 - Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides can utilize either NADP or NAD as coenzyme. The enzyme's three-dimensional structure has been solved (Rowland et al., 1994, Structure 2, 1073-1087) and shown to contain a conventional nucleotide binding domain. NADP+ was modeled into the structure by superimposing the beta alpha beta domain and that of coenzyme-bound 6-phosphogluconate dehydrogenase (Adams et al., 1994, Structure 2, 651-658), enabling us to identify Arg-46 as a potentially important residue for NADP+ binding. Using site-directed mutagenesis, we constructed mutant enzymes in which Arg-46 was replaced by glutamine (R46Q) and alanine (R46A) and examined their kinetic properties. The principal effects in these mutant enzymes were that the Km and Ki values for NADP+ increased by 2 to 3 orders of magnitude over those of the wild-type enzyme. No other kinetic constant was altered more than 6.5-fold. Changing this single amino acid leads to mutant glucose-6-phosphate dehydrogenases with coenzyme specificities that favor NAD+, whereas the wild-type enzyme prefers NADP+ as coenzyme. These results confirm that Arg-46 plays a key role in NADP+ binding by contributing a positively charged planar residue that interacts primarily with the 2'-adenosine phosphate. The Arg residue corresponding to Arg-46 in L. mesenteroides glucose-6-phosphate dehydrogenase is conserved in all glucose-6-phosphate dehydrogenases and, presumably, plays the same role in all these enzymes. SN - 0003-9861 UR - https://www.unboundmedicine.com/medline/citation/8579362/Identification_of_an_arginine_residue_in_the_dual_coenzyme_specific_glucose_6_phosphate_dehydrogenase_from_Leuconostoc_mesenteroides_that_plays_a_key_role_in_binding_NADP+_but_not_NAD+_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0003-9861(96)90058-2 DB - PRIME DP - Unbound Medicine ER -