Intrinsic stability and extrinsic stabilization of creatinase from Pseudomonas putida

• Published in: Biological chemistry Hoppe-Seyler Vol. 374; no. 7; p. 427
• Main Authors: Schumann, J, Möllering, H, Jaenicke, R
• Format: Journal Article
• Language: English
• Published: Germany 01-07-1993
• Subjects: Circular Dichroism; Cloning, Molecular; Escherichia coli - genetics; Glycerol - chemistry; Oxidation-Reduction; Protein Denaturation; Pseudomonas putida - enzymology; Pseudomonas putida - genetics; Recombinant Proteins - biosynthesis; Spectrometry, Fluorescence; Temperature; Ureohydrolases - biosynthesis; Ureohydrolases - chemistry; Ureohydrolases - isolation & purification
• ISSN: 0177-3593
• DOI: 10.1515/bchm3.1993.374.7-12.427

Creatinase (creatine amidinohydrolase, EC 3.5.3.3), a homodimer of 45 kDa subunit molecular mass, shows only limited functional stability, and is inaccessible to reconstitution after preceding deactivation, denaturation and dissociation. The enzyme has been characterized regarding its native and denatured states. Studying its unfolding characteristics in the presence of "extrinsic factors", such as DTE, BSA and glycerol, it was possible to define solvent conditions where the stability of the enzyme is significantly improved. Apart from protecting essential thiol groups and charge screening effects, the stabilization is caused mainly by preferential solvation. In the presence of 20% (w/v) glycerol, the kinetic analysis of the time course of denaturation indicates that a partially active folding intermediate, rather than the whole molecule, is involved in the stabilization. The mixed solvent improves the thermal stability, as well as the stability toward GdmCl and urea.