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Effect of limited proteolysis on the stability and enzymatic activity of human placental S ‐adenosylhomocysteine hydrolase
Author(s) -
Huang He,
Yuan ChongSheng,
Borchardt Ronald T.
Publication year - 1997
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560060712
Subject(s) - chemistry , enzyme , hydrolase , guanidine , tryptophan , proteolysis , papain , protein tertiary structure , trypsin , native state , biochemistry , enzyme assay , circular dichroism , stereochemistry , amino acid
Abstract Human placental S ‐adenosylhomocysteine (AdoHcy) hydrolase was subjected to limited papain digestion. The multiple cleavage sites in the enzyme were identified to be Lys 94 —Ala 95 , Tyr 100 ‐Ala 101 , Glu 243 ‐Ile 244 , Met 367 ‐Ala 368 , Gln 369 ‐Ile 370 , and Gly 382 ‐Val 383 . Despite multiple cleavage sites in the backbone of the protein, the digested enzyme was able to maintain its quaternary structure and retain its full catalytic activity. The enzyme activity of the partially digested AdoHcy hydrolase was essentially identical to that of the native enzyme at several pH values. The thermal stabilities of the native and partially digested enzymes were only slightly different at all temperatures tested. The stability of both native and partially digested enzymes were examined in guanidine hydrochloride and equilibrium unfolding transitions were monitored by CD spectroscopy and tryptophan fluorescence spectroscopy. The results of these experiments can be summarized as follows: (1) CD spectroscopic analysis showed that the overall secondary and tertiary structures of the partially digested enzyme are essentially identical with those of the native enzyme; and (2) tryptophan fluorescence spectroscopic analysis indicated that there are small differences in the environments of surface‐exposed tryptophan residues between the partially digested enzyme and the native enzyme under unfolding conditions. The differences in the free energy of unfolding, Δ(Δ G u ) [Δ G u (native)—Δ G u (digested)], is approximately 1.3 kcal/mol. When NAD + was removed from the partially digested enzyme, the secondary and tertiary structures of the apo form of the digested AdoHcy hydrolase were completely lost and the enzymatic activity could not be recovered by incubation with excess NAD + . These results suggest that AdoHcy hydrolase exists as a very compact enzyme with extensive intramolecular bonding, which contributes significantly to the overall global protein stabilization. Identification of the surface‐exposed peptide bonds, which are susceptible to papain digestion, has provided some constraints on the spatial orientations of subunits of the enzyme. This information, in turn, has provided supplemental data for X‐ray crystallographic studies currently ongoing in our laboratories.