Function of threonine-55 in the carbamoyl phosphate binding site of Escherichia coli aspartate transcarbamoylase

Biochemistry ◽  
1989 ◽  
Vol 28 (26) ◽  
pp. 9937-9943 ◽  
Author(s):  
Wei Xu ◽  
Evan R. Kantrowitz
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Aspartate Transcarbamoylase ◽  
Phosphate Binding ◽  
Carbamoyl Phosphate

scholarly journals Aspartate transcarbamoylase from Phaseolus aureus. Partial purification and properties

1972 ◽  
Vol 129 (3) ◽  
pp. 571-581 ◽  
Author(s):  
B. L. Ong ◽  
J. F. Jackson
Keyword(s):  
Gel Filtration ◽  
Deae Cellulose ◽  
Aspartate Transcarbamoylase ◽  
Partial Purification ◽  
Saturation Curve ◽  
Phaseolus Aureus ◽  
Phosphate Binding ◽  
Carbamoyl Phosphate ◽  
Sequential Reaction ◽  
Substrate Saturation

1. Aspartate transcarbamoylase from 4-day-old radicles of Phaseolus aureus was purified 190-fold by (NH4)2SO4 fractionation, DEAE-cellulose and DEAE-Sephadex chromatography and Sephadex-gel filtration. The partially purified enzyme, which required Pi for maximum stability, had an apparent molecular weight of 83000±5000. 2. Uridine nucleotides were found to inhibit the activity; UMP was the most potent inhibitor, followed by UDP and UTP. No other nucleotide was found to affect the enzyme, nor could UMP inhibition be overcome by adding another nucleotide. Aspartate gives a hyperbolic substrate-saturation curve, both with and without UMP. The nucleotide inhibitor is non-competitive with respect to this substrate. Carbamoyl phosphate also yields a hyperbolic substrate-saturation curve in the absence of feedback inhibitor, but when UMP is added a sigmoidal pattern results, and the inhibition is competitive with carbamoyl phosphate. 3. The degree of inhibition by UMP is not affected by p-chloromercuribenzoate, urea, mild heat pretreatment or change in pH over the range 8.5–10.5, but is affected by temperature. 4. The aspartate analogue, succinate, both activates and inhibits the reaction, depending on the concentrations of aspartate and succinate used. 5. Kinetic studies with the partially purified enzyme showed that the Km for carbamoyl phosphate (0.091 mm) is much lower than that for aspartate (1.7mm). A sequential reaction mechanism was inferred from product-inhibition kinetics, with carbamoyl phosphate binding to the enzyme before aspartate, and the product, carbamoylaspartate, being released ahead of Pi. Initial-velocity studies gave a set of parallel reciprocal plots, compatible with an essentially irreversible step occurring before the binding of aspartate.

scholarly journals Structure of l-Xylulose-5-Phosphate 3-Epimerase (UlaE) from the Anaerobic l-Ascorbate Utilization Pathway of Escherichia coli: Identification of a Novel Phosphate Binding Motif within a TIM Barrel Fold

2008 ◽  
Vol 190 (24) ◽  
pp. 8137-8144 ◽  
Author(s):  
Rong Shi ◽  
Marco Pineda ◽  
Eunice Ajamian ◽  
Qizhi Cui ◽  
Allan Matte ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Active Site ◽  
Active Sites ◽  
Triosephosphate Isomerase ◽  
Pentose Phosphate ◽  
Binding Motif ◽  
Phosphate Binding ◽  
Activity Measurements ◽  
Tim Barrel

ABSTRACT Three catabolic enzymes, UlaD, UlaE, and UlaF, are involved in a pathway leading to fermentation of l-ascorbate under anaerobic conditions. UlaD catalyzes a β-keto acid decarboxylation reaction to produce l-xylulose-5-phosphate, which undergoes successive epimerization reactions with UlaE (l-xylulose-5-phosphate 3-epimerase) and UlaF (l-ribulose-5-phosphate 4-epimerase), yielding d-xylulose-5-phosphate, an intermediate in the pentose phosphate pathway. We describe here crystallographic studies of UlaE from Escherichia coli O157:H7 that complete the structural characterization of this pathway. UlaE has a triosephosphate isomerase (TIM) barrel fold and forms dimers. The active site is located at the C-terminal ends of the parallel β-strands. The enzyme binds Zn2+, which is coordinated by Glu155, Asp185, His211, and Glu251. We identified a phosphate-binding site formed by residues from the β1/α1 loop and α3′ helix in the N-terminal region. This site differs from the well-characterized phosphate-binding motif found in several TIM barrel superfamilies that is located at strands β7 and β8. The intrinsic flexibility of the active site region is reflected by two different conformations of loops forming part of the substrate-binding site. Based on computational docking of the l-xylulose 5-phosphate substrate to UlaE and structural similarities of the active site of this enzyme to the active sites of other epimerases, a metal-dependent epimerization mechanism for UlaE is proposed, and Glu155 and Glu251 are implicated as catalytic residues. Mutation and activity measurements for structurally equivalent residues in related epimerases supported this mechanistic proposal.

Biodegradative ornithine decarboxylase of Escherichia coli. Purification, properties, and pyridoxal 5'-phosphate binding site

Biochemistry ◽  
1975 ◽  
Vol 14 (16) ◽  
pp. 3675-3681 ◽  
Author(s):  
Deborah Applebaum ◽  
Donna L. Sabo ◽  
Edmond H. Fischer ◽  
David R. Morris
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Ornithine Decarboxylase ◽  
Phosphate Binding
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