Cellular Retinoid-Binding Proteins: Limited Proteolysis Reveals a Conformational Change upon Ligand Binding

Biochemistry ◽  
1994 ◽  
Vol 33 (10) ◽  
pp. 2873-2879 ◽  
Author(s):  
Robert S. Jamison ◽  
Marcia E. Newcomer ◽  
David E. Ong
Keyword(s):  
Ligand Binding ◽  
Conformational Change ◽  
Binding Proteins ◽  
Limited Proteolysis ◽  
Retinoid Binding Proteins

scholarly journals Ligand binding on to maize (Zea mays) malate synthase: a structural study

1994 ◽  
Vol 303 (2) ◽  
pp. 413-421 ◽  
Author(s):  
S Beeckmans ◽  
A S Khan ◽  
L Kanarek ◽  
E Van Driessche
Keyword(s):  
Zea Mays ◽  
Ligand Binding ◽  
Conformational Change ◽  
Limited Proteolysis ◽  
Enzymic Activity ◽  
Malate Synthase ◽  
Acetyl Coa ◽  
Original Activity ◽  
Kinetic Measurements ◽  
Michaelis Constants

A kinetic and ligand binding study on maize (Zea mays) malate synthase is presented. It is concluded from kinetic measurements that the enzyme proceeds through a ternary-complex mechanism. Michaelis constants (Km,glyoxylate and Km,acetyl-CoA) were determined to be 104 microM and 20 microM respectively. C.d. measurements in the near u.v.-region indicate that a conformational change is induced in the enzyme by its substrate, glyoxylate. From these studies we are able to calculate the affinity for the substrate (Kd,glyoxylate) as 100 microM. A number of inhibitors apparently trigger the same conformational change in the enzyme, i.e. pyruvate, glycollate and fluoroacetate. Another series of inhibitors bearing more bulky groups and/or an extra carboxylic acid also induce a conformational change, which is, however, clearly different from the former one. Limited proteolysis with trypsin results in cleavage of malate synthase into two fragments of respectively 45 and 19 kDa. Even when no more intact malate synthase chains are present, the final enzymic activity still amounts to 30% of the original activity. If trypsinolysis is performed in the presence of acetyl-CoA, the cleavage reaction is appreciably slowed down. The dissociation constant for acetyl-CoA (Kd,acetyl-CoA) was calculated to be 14.8 microM when the glyoxylate subsite is fully occupied by pyruvate and 950 microM (= 50 x Km) when the second subsite is empty. It is concluded that malate synthase follows a compulsory-order mechanism, glyoxylate being the first-binding substrate. Glyoxylate triggers a conformational change in the enzyme and, as a consequence, the correctly shaped binding site for acetyl-CoA is created. Demetallization of malate synthase has no effect on the c.d. spectrum in the near u.v.-region. Moreover, glyoxylate induces the same spectral change in the absence of Mg2+ as in its presence. Nevertheless, malate synthase shows no activity in the absence of the cation. We conclude that Mg2+ is essential for catalysis, rather than for the structure of the enzyme's catalytic site.

scholarly journals Diversity in the structures and ligand-binding sites of nematode fatty acid and retinol-binding proteins revealed by Na-FAR-1 from Necator americanus

2015 ◽  
Vol 471 (3) ◽  
pp. 403-414 ◽  
Author(s):  
M. Florencia Rey-Burusco ◽  
Marina Ibáñez-Shimabukuro ◽  
Mads Gabrielsen ◽  
Gisela R. Franchini ◽  
Andrew J. Roe ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Ligand Binding ◽  
Tissue Distribution ◽  
Binding Sites ◽  
Binding Proteins ◽  
Retinol Binding Protein ◽  
Internal Cavity ◽  
Binding Characteristics ◽  
Necator Americanus ◽  
Ligand Binding Sites

Necator americanus fatty acid and retinol-binding protein-1 (Na-FAR-1) is an abundantly expressed FAR from a parasitic hookworm. The present work describes its tissue distribution, structure and ligand-binding characteristics and shows that Na-FAR-1 expands to transport multiple FA molecules in its internal cavity.

scholarly journals Crystal structures of PI3K-C2α PX domain indicate conformational change associated with ligand binding

2008 ◽  
Vol 8 (1) ◽  
pp. 13 ◽  
Author(s):  
Gary N Parkinson ◽  
David Vines ◽  
Paul C Driscoll ◽  
Snezana Djordjevic
Keyword(s):  
Ligand Binding ◽  
Crystal Structures ◽  
Conformational Change ◽  
Px Domain
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