The Effect of Valine Substitution for Glycine in the Dimer Interface of Citrate Synthase from Thermoplasma acidophilum on Stability and Activity

• Published in: Biochemical and biophysical research communications Vol. 275; no. 2; pp. 460 - 465
• Main Authors: Kocabiyik, S, Erduran, I
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 28-08-2000
• Subjects: Base Sequence; Citrate (si)-Synthase - chemistry; Citrate (si)-Synthase - genetics; Citrate (si)-Synthase - metabolism; citrate synthase; DNA Primers; Enzyme Stability; Glycine - chemistry; Glycine - metabolism; hydrophobic interactions; Mutagenesis, Site-Directed; Protein Denaturation; site-directed mutagenesis; Thermoplasma - enzymology; Thermoplasma acidophilum; thermostability; Valine - chemistry; Valine - metabolism; citrate synthase; site-directed mutagenesis; Thermoplasma acidophilum; hydrophobic interactions; thermostability
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1006/bbrc.2000.3310

To determine the role of hydrophobic interactions in the dimer interface of citrate synthase (CS) from Thermoplasma (Tp) acidophilum in thermostabilization, we have used site-directed mutagenesis to replace Gly 196 by Val on the helix L of the subunit interface. Recombinant wild-type and Gly 196 mutant TpCS enzymes were largely identical in terms of substrate specificities (Km for oxaloacetate and acetyl CoA). However, the mutation not only reduced catalytic activity (about 10-fold) (i.e., Vmax, kcat and specific activity) of the TpCS, but also decreased its thermal and chemical stability. Archaeal citrate synthase is active as a dimer, since residues from both monomers participate in the active site. Our results suggest that Gly196 → Val mutation interferes with dimerization, so that improper dimerization or dissociation of the dimer would have a profound affect on the activity as well as the conformational stability of TpCS.