Related Experiment Video

Updated: Sep 26, 2026

Functional Complementation Analysis (FCA): A Laboratory Exercise Designed and Implemented to Supplement the Teaching of Biochemical Pathways
09:27

Functional Complementation Analysis (FCA): A Laboratory Exercise Designed and Implemented to Supplement the Teaching of Biochemical Pathways

Published on: June 24, 2016

Amino acids Glu323, Tyr324, Glu330, and Val331 of factor Va heavy chain are essential for expression of cofactor

Lisam S Singh1, Michael A Bukys, Daniel O Beck

  • 1Department of Chemistry, Cleveland State University, Cleveland, Ohio 44115, USA.

The Journal of Biological Chemistry
|May 10, 2003
PubMed

We have recently demonstrated that amino acid region 323-331 of factor Va heavy chain (9 amino acids, AP4') contains a binding site for factor Xa (Kalafatis, M., and Beck, D. O. (2002) Biochemistry 41, 12715-12728). To ascertain which amino acids within this region are important for the effector and receptor properties of the cofactor with respect to factor Xa, we have synthesized three overlapping peptides (5 amino acids each) spanning the amino acid region 323-331 and tested them for their effect on prothrombinase complex assembly and function. Peptide containing amino acids 323EYFIA327 alone was found to increase the catalytic efficiency of factor Xa but had no effect on the fluorescent anisotropy of active site-labeled factor Xa (human factor Xa labeled in the active site with Oregon Green 488; [OG488]-EGR-hXa). In contrast, peptide containing the sequence 327AAEEV331 was found to interact with [OG488]-EGR-hXa with half-maximal saturation reached at approximately 150 microm, but it was unable to produce a cofactor effect on factor Xa. Peptide 325FIAAE329 inhibited prothrombinase activity and was able to partially decrease the fluorescent anisotropy of [OG488]-EGR-hXa but could not increase the catalytic efficiency of factor Xa with respect to prothrombin. A control peptide with the sequence FFFIA did not increase the catalytic efficiency of factor Xa, whereas a peptide with the sequence AAEMI was impaired in its capability to interact with [OG488]-EGR-hXa. Two mutant recombinant factor Va molecules (Glu323 --> Phe/Tyr324 --> Phe, factor VaFF; Glu330 --> Met/Val331 --> Ile, factor VaMI) showed impaired cofactor activity when used at limiting cofactor concentration, whereas the quadruple mutant (Glu323 --> Phe/Tyr324 --> Phe and Glu330 --> Met/Val331 --> Ile, factor VaFF/MI) had no cofactor activity under similar experimental conditions. Our data demonstrate that amino acid residues Glu323, Tyr324, Glu330, and Val331 of factor Va heavy chain are critical for expression of factor Va cofactor activity.

More Related Videos

Homogeneous Glycoconjugate Produced by Combined Unnatural Amino Acid Incorporation and Click-Chemistry for Vaccine Purposes13:53

Homogeneous Glycoconjugate Produced by Combined Unnatural Amino Acid Incorporation and Click-Chemistry for Vaccine Purposes

Published on: December 19, 2020

Optimized Protocol for the Extraction of Proteins from the Human Mitral Valve09:13

Optimized Protocol for the Extraction of Proteins from the Human Mitral Valve

Published on: June 14, 2017

Related Concept Videos

Cofactors and Coenzymes01:24

Cofactors and Coenzymes

Enzymes are proteins made of amino acids. The functional group of each constituent amino acid catalyzes a wide variety of chemical reactions via ionic interactions or acid-base reactions. However, amino acids cannot catalyze oxidation-reduction and group transfer reactions and need to be aided by non-protein components called cofactors. Cofactors are also referred to as the chemical teeth of an enzyme.
Cofactors can be metallic ions or organic molecules called coenzymes. These types of helper...
Cofactors and Coenzymes01:27

Cofactors and Coenzymes

Enzymes require additional components for proper function. There are two such classes of molecules: cofactors and coenzymes. Cofactors are metallic ions and coenzymes are non-protein organic molecules. Both of these types of helper molecule can be tightly bound to the enzyme or bound only when the substrate binds.
Cofactors and Coenzymes01:27

Cofactors and Coenzymes

Enzymes require additional components for proper function. There are two such classes of molecules: cofactors and coenzymes. Cofactors are metallic ions and coenzymes are non-protein organic molecules. Both of these types of helper molecule can be tightly bound to the enzyme or bound only when the substrate binds.
tRNA Activation02:26

tRNA Activation

Aminoacyl-tRNA synthetases are present in both eukaryotes and bacteria. Though eukaryotes have 20 different aminoacyl-tRNA synthetases to couple to 20 amino acids, many bacteria do not have genes for all of these aminoacyl-tRNA synthetases. Despite this, they still use all 20 amino acids to synthesize their proteins. For instance, some bacteria do not have the gene encoding the enzyme that couples glutamine with its partner tRNA. In these organisms, one enzyme adds glutamic acid to all of the...
tRNA Activation02:26

tRNA Activation

Aminoacyl-tRNA synthetases are present in both eukaryotes and bacteria. Though eukaryotes have 20 different aminoacyl-tRNA synthetases to couple to 20 amino acids, many bacteria do not have genes for all of these aminoacyl-tRNA synthetases. Despite this, they still use all 20 amino acids to synthesize their proteins. For instance, some bacteria do not have the gene encoding the enzyme that couples glutamine with its partner tRNA. In these organisms, one enzyme adds glutamic acid to all of the...
Amino Acid Catabolism01:18

Amino Acid Catabolism

Microorganisms rely on proteins as an essential carbon and energy source, particularly in environments with limited polysaccharides or lipids. However, proteins are too large to cross the plasma membrane unaided, necessitating enzymatic degradation. Microbes secrete extracellular proteases and peptidases that hydrolyze proteins into peptides, which can then be transported across the membrane. Once inside the cell, intracellular proteases degrade these peptides into free amino acids, which...