Several aspects of the properties of yeast invertase will be investigated to determine the role that carbohydrate (CHO) may play in influencing this glycoprotein's structure and function. To obtain invertase with varying amounts of CHO, external invertase whose mass consists of 50 percent CHO, will be deglycosylated with Endo H and Alpha-mannosidase for specified periods of time. Invertases from yeast mutants with varying amounts of CHO are also available. Yeast internal invertase will be used as an example of a form of this enzyme which contains no carbohydrate at all. To establish whether CHO content can influence enzyme activity, these various forms of invertase will be compared for their activity loss with time of incubation at pH 4.0, susceptibility to proteolysis and ability to recover activity following denaturation. To determine if conformational differences exist between the glycosylated and deglycosylated forms of invertase, CD, UV, and fluorescence difference spectra will be employed, as will deuterium exchange studies. The relationship between enzyme activity and degree of subunit aggregation will also be examined to determine whether a correlation exists between the two. To complement these studies, an analysis of the complete amino acid sequence of external invertase will be undertaken, and a comparison of the amino acid content, end groups, and peptide and fingerprint profiles of external and internal invertases will be made to firmly establish that these two proteins are identical. This study should also establish the location of the nine oligosaccharide chains in external invertase and whether those CHO chains which are inaccessible to Endo H differ in size from those that are accessible. Group specific reagents will be used to determine the effect that modifying specific amino acids has on enzyme activity. In an attempt to identify the active site region in invertase, derivatives of sucrose or raffinose are planned which bind covalently to this region.
