Structure and reactivity relationship in glyceraldehyde-3-phosphate dehydrogenase. Dinitrophenylation of cysteine residues of yeast and rabbit muscle enzymes.
Dinitrophenylation of rabbit muscle and yeast glyceraldehyde-3-phosphate dehydrogenases modifies only SH groups. The rabbit muscle apoenzyme loses 75% of its original activity upon dinitrophenylation of two SH groups per tetramer whereas the yeast apoenzyme is totally inactivated under the same conditions. Dinitrophenylation of the active-site cysteine-149 of rabbit muscle and yeast holoenzymes results in an loss of activity corresponding to a 'half-of-the-sites' and a 'full-sites' reactivity, respectively. Determination of the sulphydryl content of the modified enzymes shows an unmasking of the cysteine residues of the dinitrophenylated rabbit muscle apoenzyme which is not observed for the yeast protein. However, conformational changes are revealed for both dinitrophenylated apoenzymes by differential absorption spectroscopy or by limited proteolysis. Sulphydryl group unmasking is not observed after modification of the cysteine residues of the rabbit muscle holoenzyme but it does occur when dinitrophenylation is performed in the presence of two moles NAD+/mole rabbit muscle enzyme. Although the apoenzyme is sensitive to an induced conformational change, our results favour symmetrical structures for both yeast apo and holo enzymes. The bis-dinitrophenylated rabbit muscle apoenzyme presents all the characteristics of an asymmetrical structure; however, it is not possible to deduce whether this symmetry is due to the chemical modification or whether it preexists in the native apoenzyme. The results of the dinitrophenylation of the rabbit holoenzyme, however, indicate that this enzyme possesses an asymmetrical structure.
D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D003545:Cysteine; D004136:Dinitrobenzenes; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D009132:Muscles / Q000201:enzymology*; D011487:Protein Conformation; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013053:Spectrophotometry; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000032:analysis
D004136:Dinitrobenzenes; D013438:Sulfhydryl Compounds; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D003545:Cysteine