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Conformational motility in D-glyceraldehyde-3-phosphate dehydrogenase influenced by subunit interactions
Abstract:
Binding of four molecules of NAD to pig muscle glyceraldehyde-3-phosphate dehydrogenase decreases the apparent reactivity of Cys-153 -- a residue exposed only temporarily -- towards PMB in all four subunits of the enzyme. However, the change of reactivity is not a linear function of the degree of saturation with coenzyme, inasmuch as the first two, tightly bound NAD's exert a much larger effect than do the other two. The apparent reactivity of Cys-153 was investigated in GAPD's produced by hybridization of enzymes modified on residue Cys-149 with different reagents. The homotetramer-NAD complexes of these modified species were shown to exhibit different dissociation constants: native GAPD less than (alkylated--Cys-149)-GAPD less than (mercaptidated--Cys-149)-GAPD. The tight binding of 2 NAD's on the hybrid tetramers decreases the reactivity to the same extent in liganded and non-liganded subunits. The decrease in the apparent reactivity of Cys-153 is due to the restriction of local conformational motility around thes residue. Binding of NAD shifts the equilibrium towards a more closed form of the protein, whereas the rate constant of mercaptide formation itself remains unaltered. These findings suggest that the NAD-induced conformational changes are also reflected in the local fluctuation of the protein around Cys-153. The subunit interactions still operate in the hybrids and mediate the NAD-induced conformational changes.
Insights
Cofactor binding to glyceraldehyde-3-phosphate dehydrogenase (GAPD) alters enzyme reactivity. NAD binding restricts conformational flexibility around Cys-153, impacting enzyme function.
Area of Science:
- Biochemistry
- Enzyme kinetics
- Protein conformational changes
Background:
- Glyceraldehyde-3-phosphate dehydrogenase (GAPD) is a key enzyme in glycolysis.
- The reactivity of cysteine residues in GAPD is crucial for its catalytic activity.
- NAD binding influences the enzyme's structure and function.
Purpose of the Study:
- To investigate the effect of NAD binding on the reactivity of Cys-153 in pig muscle GAPD.
- To explore how coenzyme saturation affects enzyme conformation and Cys-153 reactivity.
- To understand the role of subunit interactions in NAD-induced conformational changes.
Main Methods:
- Enzyme kinetics studies using pig muscle GAPD.
- Investigation of Cys-153 reactivity towards PMB.
- Hybridization of GAPD subunits modified at Cys-149.
- Determination of dissociation constants for NAD binding to modified GAPD species.
Main Results:
- NAD binding to GAPD decreases the apparent reactivity of Cys-153 in all subunits.
- The effect of NAD binding is non-linear, with the first two NAD molecules having a greater impact.
- Hybrid GAPD species showed altered NAD dissociation constants.
- NAD binding restricts local conformational motility around Cys-153, favoring a closed protein conformation.
Conclusions:
- NAD-induced conformational changes in GAPD are reflected in local fluctuations around Cys-153.
- Subunit interactions mediate these NAD-induced conformational changes.
- The observed decrease in Cys-153 reactivity is attributed to restricted conformational flexibility.