N-(5-Phosphoribosyl)anthranilate isomerase-indoleglycerol-phosphate synthase. 2. Fast-reaction studies show that a fluorescent substrate analog binds independently to two different sites

Biochemistry ◽  
1979 ◽  
Vol 18 (26) ◽  
pp. 5953-5959 ◽  
Author(s):  
Willy Cohn ◽  
Kasper Kirschner ◽  
Carlton Paul
Keyword(s):  
Fast Reaction ◽  
Fluorescent Substrate ◽  
Substrate Analog ◽  
Phosphate Synthase

scholarly journals Structure and kinetics of indole-3-glycerol phosphate synthase from Pseudomonas aeruginosa: Decarboxylation is not essential for indole formation

2020 ◽  
Vol 295 (47) ◽  
pp. 15948-15956
Author(s):  
Annika Söderholm ◽  
Matilda S. Newton ◽  
Wayne M. Patrick ◽  
Maria Selmer
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Phenyl Group ◽  
Nucleophilic Attack ◽  
Active Site Residues ◽  
Glycerol Phosphate ◽  
E Coli ◽  
Substrate Analog ◽  
The 1960S ◽  
Kinetics Of ◽  
Phosphate Synthase

In tryptophan biosynthesis, the reaction catalyzed by the enzyme indole-3-glycerol phosphate synthase (IGPS) starts with a condensation step in which the substrate's carboxylated phenyl group makes a nucleophilic attack to form the pyrrole ring of the indole, followed by a decarboxylation that restores the aromaticity of the phenyl. IGPS from Pseudomonas aeruginosa has the highest turnover number of all characterized IGPS enzymes, providing an excellent model system to test the necessity of the decarboxylation step. Since the 1960s, this step has been considered to be mechanistically essential based on studies of the IGPS–phosphoribosylanthranilate isomerase fusion protein from Escherichia coli. Here, we present the crystal structure of P. aeruginosa IGPS in complex with reduced CdRP, a nonreactive substrate analog, and using a sensitive discontinuous assay, we demonstrate weak promiscuous activity on the decarboxylated substrate 1-(phenylamino)-1-deoxyribulose-5-phosphate, with an ∼1000× lower rate of IGP formation than from the native substrate. We also show that E. coli IGPS, at an even lower rate, can produce IGP from decarboxylated substrate. Our structure of P. aeruginosa IGPS has eight molecules in the asymmetric unit, of which seven contain ligand and one displays a previously unobserved conformation closer to the reactive state. One of the few nonconserved active-site residues, Phe201 in P. aeruginosa IGPS, is by mutagenesis demonstrated to be important for the higher turnover of this enzyme on both substrates. Our results demonstrate that despite IGPS's classification as a carboxy-lyase (i.e. decarboxylase), decarboxylation is not a completely essential step in its catalysis.

scholarly journals Fluorescent substrate analog for monitoring chain elongation by undecaprenyl pyrophosphate synthase in real time

2011 ◽  
Vol 417 (1) ◽  
pp. 136-141 ◽  
Author(s):  
Kuo-Hsun Teng ◽  
Annie P.-C. Chen ◽  
Chih-Jung Kuo ◽  
Yu-Chin Li ◽  
Hon-Ge Liu ◽  
...  
Keyword(s):  
Real Time ◽  
Chain Elongation ◽  
Fluorescent Substrate ◽  
Substrate Analog

scholarly journals Modes of action of tunicamycin, liposidomycin B, and mureidomycin A: inhibition of phospho-N-acetylmuramyl-pentapeptide translocase from Escherichia coli.

1996 ◽  
Vol 40 (7) ◽  
pp. 1640-1644 ◽  
Author(s):  
P E Brandish ◽  
K I Kimura ◽  
M Inukai ◽  
R Southgate ◽  
J T Lonsdale ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antibacterial Agents ◽  
Enzyme Assay ◽  
Molecular Mechanisms ◽  
Fluorescent Substrate ◽  
Acceptor Substrate ◽  
Nucleoside Antibiotics ◽  
Substrate Analog ◽  
Binding Inhibition ◽  
Insight Into

Using a continuous fluorescence-based enzyme assay, we have characterized the antibacterial agents tumicamycin and liposidomycin B as inhibitors of solubilized Escherichia coli phospho-N-acetylmuramyl-pentapeptide translocase. Tunicamycin exhibited reversible inhibition (Ki = 0.55 +/- 0.1 microM) which was noncompetitive with respect to the lipid acceptor substrate and competitive with respect to the fluorescent substrate analog, dansyl-UDPMurNAc-pentapeptide. Liposidomycin B exhibited slow-binding inhibition (Ki = 80 +/- 15 nM) which was competitive with respect to the lipid acceptor substrate and noncompetitive with respect to dansyl-UDPMurNAc-pentapeptide. These results provide insight into the molecular mechanisms of action of these two classes of nucleoside antibiotics.

Fluorescent substrate analog for adenosine deaminase: 3'-O-[5-(dimethylamino)naphthalene-1-sulfonyl]adenosine

Biochemistry ◽  
1981 ◽  
Vol 20 (11) ◽  
pp. 3103-3109 ◽  
Author(s):  
Gedalyahu Skorka ◽  
Pessia Shuker ◽  
David Gill ◽  
Jacob Zabicky ◽  
Abraham H. Parola
Keyword(s):  
Adenosine Deaminase ◽  
Fluorescent Substrate ◽  
Substrate Analog

PAD2 Activity Monitored via a Fluorescent Substrate Analog

2015 ◽  
Vol 86 (4) ◽  
pp. 599-605
Author(s):  
Mary J. Sabulski ◽  
Yanming Wang ◽  
Marcos M. Pires
Keyword(s):  
Fluorescent Substrate ◽  
Substrate Analog
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