PART A: STUDIES ON SOYBEAN LIPOXYGENASES. PART B: STUDIES ON TWO ALPHA-GLUCOSIDASES FROM YEAST
Abstract
A variety of chemical techniques have been used to extend our knowledge of the reaction mechanisms of lipoxygenases and glucosidases. The characteristics of the secondary reaction of lipoxygenase-3 were probed. Labeling and inhibition studies indicated the secondary reaction was independent of the dioxygenase activity. The dimeric nature of the products suggests the secondary reaction involves the formation of free radicals from linoleic-hydroperoxide. The radicals formed can diffuse from the enzyme and react in solution. Paramagnetic susceptibility measurements show the iron in native lipoxygenase-1 is oxidized from high spin iron two to high spin iron three by its product hydroperoxide. This oxidation is probably the mechanism responsible for the product activation previously reported. A series of 1-amine pentatols were found to inhibit Saccharomyces oviformis maltase as well as (beta)-glucosidase of almond and coffee bean (alpha)-galactosidase. The inhibition was selective for the enantiomeric analog of the normal substrate. The pentatolamines are weaker inhibitors than the corresponding hexosyl and pentosyl-amines, but stronger than the free sugars. Thus the presence of a positive charge near carbon one appears to be more important than possession of the intact sugar structure. However, the correct sugar structure is important for maximum efficiency. Labeling studies on maltase and (alpha)-methylglucosidase, using n-ethylmaleamide and carbodiimide suggest the presence of essential sulfhydryl and carboxyl groups are present at the active site. Amino acid analysis shows maltase contains one essential sulfhydryl group. Proteolysis and peptide isolation indicate both enzymes have two essential active site carboxyl groups.
Degree
Ph.D.
Subject Area
Biochemistry
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