Abstract
| Originalsprog | Engelsk |
|---|---|
| Tidsskrift | Archives of Biochemistry and Biophysics |
| Vol/bind | 490 |
| Udgave nummer | 1 |
| Sider (fra-til) | 42-9 |
| Antal sider | 7 |
| ISSN | 0003-9861 |
| DOI | |
| Status | Udgivet - 2009 |
Bibliografisk note
Keywords: Binding Sites; Kinetics; Magnesium; Methanococcaceae; Models, Biological; Models, Molecular; Mutation; Nucleotide Deaminases; Phosphates; Protein Binding; Protein Conformation; Protein Structure, Secondary; Pyrophosphatases; Substrate SpecificityAdgang til dokumentet
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I: Archives of Biochemistry and Biophysics, Bind 490, Nr. 1, 2009, s. 42-9.
Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › peer review
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TY - JOUR
T1 - Concerted bifunctionality of the dCTP deaminase-dUTPase from Methanocaldococcus jannaschii: a structural and pre-steady state kinetic analysis
AU - Siggaard, Julie H B
AU - Johansson, Eva
AU - Vognsen, Tina Reinholdt
AU - Helt, Signe Smedegaard
AU - Harris, Pernille
AU - Larsen, Sine
AU - Willemoës, Martin
N1 - Keywords: Binding Sites; Kinetics; Magnesium; Methanococcaceae; Models, Biological; Models, Molecular; Mutation; Nucleotide Deaminases; Phosphates; Protein Binding; Protein Conformation; Protein Structure, Secondary; Pyrophosphatases; Substrate Specificity
PY - 2009
Y1 - 2009
N2 - Two mutant dCTP deaminase-dUTPases from Methanocaldococcus jannaschii were crystallised and the crystal structures were solved: E145A in complex with the substrate analogue alpha,beta-imido-dUTP and E145Q in complex with diphosphate. Both mutant enzymes were defect in the deaminase reaction and had reduced dUTPase activity. In the structure of E145Q in complex with diphosphate, the diphosphate occupied the same position as the beta- and gamma-phosphoryls of the nucleotide analogue in the E145A complex. The C-terminal region that is unresolved in the apo-form of the enzyme was ordered in both complexes and closed over the active site by interacting with the phosphate backbone of the nucleotide or with the diphosphate. A magnesium ion was readily observed to complex with all three phosphoryls in the nucleotide complex or with the diphosphate. A water molecule that is likely to be involved in the nucleotidyl diphosphorylase reaction was observed in the E145A:alpha,beta-imido-dUTP complex and positioned similarly as in the monofunctional trimeric dUTPase. A comparison of the active sites of the bifunctional enzyme and the monofunctional family members, dCTP deaminase and dUTPase, suggests similar reaction mechanisms. The similar side chain conformations in the deaminase site between the nucleotide and diphosphate complexes indicated a concerted re-arrangement, or induced fit, of the whole active site promoted by enzyme and nucleotide phosphoryl interactions. A pre-steady state kinetic analysis of the bifunctional reaction and the dUTPase half-reaction supported a conformational change upon substrate binding in both reactions and a concerted catalytic step for the bifunctional reaction.
AB - Two mutant dCTP deaminase-dUTPases from Methanocaldococcus jannaschii were crystallised and the crystal structures were solved: E145A in complex with the substrate analogue alpha,beta-imido-dUTP and E145Q in complex with diphosphate. Both mutant enzymes were defect in the deaminase reaction and had reduced dUTPase activity. In the structure of E145Q in complex with diphosphate, the diphosphate occupied the same position as the beta- and gamma-phosphoryls of the nucleotide analogue in the E145A complex. The C-terminal region that is unresolved in the apo-form of the enzyme was ordered in both complexes and closed over the active site by interacting with the phosphate backbone of the nucleotide or with the diphosphate. A magnesium ion was readily observed to complex with all three phosphoryls in the nucleotide complex or with the diphosphate. A water molecule that is likely to be involved in the nucleotidyl diphosphorylase reaction was observed in the E145A:alpha,beta-imido-dUTP complex and positioned similarly as in the monofunctional trimeric dUTPase. A comparison of the active sites of the bifunctional enzyme and the monofunctional family members, dCTP deaminase and dUTPase, suggests similar reaction mechanisms. The similar side chain conformations in the deaminase site between the nucleotide and diphosphate complexes indicated a concerted re-arrangement, or induced fit, of the whole active site promoted by enzyme and nucleotide phosphoryl interactions. A pre-steady state kinetic analysis of the bifunctional reaction and the dUTPase half-reaction supported a conformational change upon substrate binding in both reactions and a concerted catalytic step for the bifunctional reaction.
U2 - 10.1016/j.abb.2009.08.005
DO - 10.1016/j.abb.2009.08.005
M3 - Journal article
C2 - 19683509
SN - 0003-9861
VL - 490
SP - 42
EP - 49
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
IS - 1
ER -