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Evidence that thermodynamic stability of papaya glutamine cyclase is only marginal
Author(s) -
Azarkan Mohamed,
Amrani Amina,
Zerhouni Samira,
Oberg Keith A.,
Ruysschaert JeanMarie,
Wintjens René,
Looze Yvan
Publication year - 2002
Publication title -
biopolymers
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/bip.10234
Subject(s) - chemistry , guanidine , equilibrium unfolding , molten globule , native state , denaturation (fissile materials) , protonation , chemical stability , circular dichroism , crystallography , cooperativity , proteolysis , protein folding , enzyme , biochemistry , organic chemistry , ion , nuclear chemistry
Papaya glutamine cyclase (PQC), a glycoprotein with a molecular mass of 32,980 Da, is a minor constituent of the papaya latex protein fraction. In neutral aqueous solutions, PQC adopts an all‐β conformation and exhibits high resistance to both proteolysis and denaturation. Complete unfolding of PQC requires a combination of an acidic medium and chemical denaturant such as urea or guanidine hydrochloride. The unfolding process takes place through formation of an intermediate A state that accumulates in the absence of chemical denaturants and displays all the features of a molten globule state. The different conformational states— N (native), A (acid‐inactivated), and U (unfolded)—have been characterized by means of circular dichroism measurements, fluorescence spectroscopies, Stokes radii determinations, and 8‐anilino‐1‐naphtalenesulfonic acid (ANS) binding characteristics. The unfolding pathways of the enzyme was further studied to estimate thermodynamic parameters characterizing both transitions N ⇔ A and A ⇔ U . In its A state, PQC is catalytically inefficient and highly susceptible to proteolysis. Also, its thermodynamic stability is decreased by some 3–5 kcal/mol. Conversion of the native to the A state involves digging up of five amino functions together with protonation of four to five acidic groups with p K a s, in the native state, around 2.7. It proceeds both cooperatively and reversibly although, in vitro, the refolding process is slow. Unfolding of the A state, on the other hand, occurs with a low degree of cooperativity. The intermediate A state thus seems to be only marginally more stable than the unfolded state. The role of suspected internal ion pairs in the stabilization of the native state of this enzyme is discussed. © 2002 Wiley Periodicals, Inc. Biopolymers 65: 325–335, 2002