FORMATION OF INTERSUBUNIT DISULFIDE BONDS AND PROPERTIES OF THE SINGLE HISTIDINE AND CYSTEINE RESIDUES IN EACH SUBUNIT RELATIVE TO THE DECAMERIC STRUCTURE OF CYANASE

Reaction of the single cysteine residue in each subunit of cyanase wit h certain SH reagents gives an active decameric derivative that dissoc iates reversibly to an inactive dimer derivative (Anderson, P. M., Joh nson, W. V., Korte, J. J., Xiong, X., Sung, Y.-c., and Fuchs, J. A. (1 988) J. Biol. Chem. 263, 5674-5680). Reaction of mixed disulfide dimer derivatives of cyanase with dithiothreitol at 0 degrees C results in formation of a disulfide bond between the subunits in the dimer. The d isulfide dimer was inactive and did not associate to a decamer; the in tersubunit disulfide bond could not be formed when the dimers were ass ociated as a decamer. The two SH groups apparently are in close proxim ity to each other in the dissociated dimer but not when the dimer is a ssociated to a decamer. Substitution of glycine for the cysteine resid ue or of tyrosine, asparagine, glycine, valine, or leucine for the sin gle histidine residue in each subunit gave mutant enzymes that were ac tive. However, H113N, H113Y, and C83G were unstable at low temperature and/or ionic strength, dissociating reversibly to an inactive dimer. Efficient reassociation required the presence of bicarbonate or cyanat e analog. The results are consistent with a proposed single site per s ubunit model explaining apparent half site binding of substrates and t he requirement of decameric structure for activity.