Neurospora crassa invertase. A study of amino acids at the active center.
1. The effects on Neurospora crassa invertase (beta-D-fructofuranoside fructohydrolase, EC 3.2.1.26) of a variety of group specific reagnets and other potential inhibitors were determined during a search for an irreversible inhibitor of the enzyme. Aniline, pyridoxal, enzyme substrate and products did not inactivate invertase under reducing conditions. Bromoacetic acid, iodoacetic acid, iodoacetamide, p-chloromercuribenzoate, hydroxylamine and 2-hydroxy-5-nitrobenzyl bromide were also ineffective. Iodine was the only reagent which irreversibly inhibited invertase. 2. Invertase was rapidly inactivated by low concentrations of iodine, indicating specific inhibition. However, the enzyme could not be protected from this inactivation by substrate. It was not reactivated by mercaptoethanol or cysteine. 3. Experiments on the uptake of radioactive iodine demonstrated that invertase is not iodinated under the conditions of iodine inactivation. 4. The sedimentation (S20,w) value of invertase was not altered by iodine inactivation. One-dimensional electrophoresis and finger-printing of tryptic digests revealed no differences between iodine treated and untreated invertase. There was no loss of carbohydrate from this glycoprotein during iodine inactivation. 5. Standard amino acid analyses of iodine-inactivated invertase showed some loss of tyrosine and a trace amount of methionine sulfone. Attempts to demonstrate oxidation of methionine to the sulfone, through modification of the procedure for preparation of samples for analysis, were unsuccessful. However, oxidation of half-cystine was indicated and further loss of tyrosine noted. A hypothesis is advanced that half-cystine is oxidized by iodine to a normally unstable oxidation state which is maintained and protected by its protein invironment and that loss of tyrosine may be an artifact caused by the presence of this residue during acid hydrolysis.
D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D002729:Chloromercuribenzoates / Q000494:pharmacology; D003545:Cysteine / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000201:enzymology*; D010446:Peptide Fragments / Q000032:analysis; D013393:Sucrase / Q000032:analysis* / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000494:pharmacology
D000596:Amino Acids; D002729:Chloromercuribenzoates; D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; D013393:Sucrase; D003545:Cysteine