Kinetic and structural studies on rabbit liver arylsulfatase A
Abstract
Arylsulfatase A is an acid hydrolase of lysosomal origin which catalyzes the hydrolysis of a variety of aryl and alkyl sulfate esters. An improved procedure for isolation of arylsulfatase A from rabbit liver is presented. The general characteristics of rabbit liver arylsulfatase A (RLASA) are also described. The inhibition constants of various inorganic anions and transition metal oxyanions for RLASA have been determined at different pH values. The pH dependence of the K$\sb{\rm i}$ values indicates that the inhibitors bind to the enzyme as dianions. The order of inhibition at pH 5.0 was sulfite $>$ vanadate $>$ phosphate $>$ tungstate $\simeq$ molybdate $>$ sulfate $>$ chromate. The potent inhibition by sulfite and by transition metal oxyanions is discussed from the viewpoint that these anions are possible transition state analogs. Phenol has little effect on the inhibition by metal oxyanions, suggesting a poor recognition of the phenyl portion of the substrate by the enzyme. It is concluded that the metal ions, aluminum and beryllium, form fluoride complexes that are responsible for the strong inhibition of RLASA. The probable inhibitory species are suggested. Based on the chemical modification studies, it is concluded that lysine and arginine are not essential for the activity of RLASA. RLASA monomer (composed of two equivalent polypeptide chains) does not contain free sulfhydryl groups but contains ten disulfide bonds. Of those, two are shown to be intersubunit disulfides. They are exposed and can be selectively sulfitolysed. The resulting sulfonated enzyme had little enzymatic activity. The generation of activity was accomplished by treatment with DTT and then autoxidation by air. The destruction of an essential histidyl residue may be responsible for an irreversible inactivation that resulted upon irradiation in the presence of vanadate. Native RLASA is shown to be particularly resistant towards proteolysis. The proteinase digestion patterns indicate that RLASA consists of two regions: a large, proteinase-resistant region constituting the N-terminal portion of the polypeptide and a proteinase-vulnerable C-terminal region. Direct sequencing of native RLASA indicated that the N-terminal end of RLASA is not blocked. Over one-third of the protein sequence was determined from peptides obtained by Lys-C and papain digestions as well as by CNBr cleavage. A putative N-glycosylation site was identified. Extensive sequence identity is observed with human ASA sequence. It is proposed that the putative subunit contact region of the RLASA monomer, which consists of two identical subunits, may involve a leucine zipper motif. The activity of the turnover-modified, inactive RLASA is partially regained upon treatment with DTT. It is suggested that the turnover modification of RLASA may involve the cleavage of the intersubunit disulfides by the SO$\sb3$-like leaving group produced during catalysis.
Degree
Ph.D.
Advisors
Etten, Purdue University.
Subject Area
Biochemistry
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