Interaction of Sheep Liver Apo-serine Hydroxymethyltransferase with Pyridoxal-5′-phosphate: A Physicochemical, Kinetic, and Thermodynamic Study

• Published in: Archives of biochemistry and biophysics 1996-06, Vol.330 (2), p.363-372
• Main Authors: Brahatheeswaran, Bhaskar, Prakash, V., Savithri, Handanahal S., Rao, N.Appaji
• Format: Article
• Language: English
• Subjects: ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; Animals; apo-serine hydroxymethyltransferase; Apoenzymes - chemistry; Apoenzymes - metabolism; CALOR; CHALEUR; Chemical Phenomena; Chemistry, Physical; COENZIMAS; COENZYME; dissociation; FOIE; Glycine Hydroxymethyltransferase - chemistry; Glycine Hydroxymethyltransferase - metabolism; HIGADO; In Vitro Techniques; internal aldimine; Kinetics; LIPOPROTEINAS; LIPOPROTEINE; Liver - enzymology; Molecular Structure; Molecular Weight; OVIN; OVINOS; PLP interaction; Protein Conformation; Protein Denaturation; Pyridoxal Phosphate - chemistry; Pyridoxal Phosphate - metabolism; RESISTANCE A LA TEMPERATURE; RESISTENCIA A LA TEMPERATURA; SERINA; SERINE; Sheep; Thermodynamics; TRANSFERASAS; TRANSFERASE
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1006/abbi.1996.0263

Sheep liver serine hydroxymethyltransferase (EC 2.1.2.1) is a homotetramer ofMr213,000 requiring pyridoxal-5′-phosphate (PLP) as cofactor. Removal of PLP from the holoenzyme converted the enzyme to the apo form which, in addition to being inactive, was devoid of the characteristic absorption spectrum. Upon the addition of PLP to the apoenzyme, complete activity was restored and the visible absorption spectrum with a maximum at 425 nm was regained. The interaction of PLP with the apoenzyme revealed two phases of reaction with pseudo-first-order rate constants of 20 ± 5 s−1and 12.2 ± 2.0 × 10−3s−1, respectively. However, addition of PLP to the apoenzyme did not cause gross conformational changes as evidenced by circular dichroic and fluorescence spectroscopy. Although conformationally apoenzyme and holoenzyme were indistinguishable, they had distinct apparent melting temperatures of 51 ± 2 and 58 ± 2°C, respectively, and the reconstituted holoenzyme was thermally as stable as the native holoenzyme. These results suggested that there was no apparent difference in the secondary structure of holoenzyme, apoenzyme, and reconstituted holoenzyme. However, sedimentation analysis of the apoenzyme revealed the presence of two peaks ofS20,wvalues of 8.7 ± 0.5 and 5.7 ± 0.3 S, respectively. A similar pattern was observed when the apoenzyme was chromatographed on a calibrated Sephadex G-150 column. The first peak corresponded to the tetrameric form (Mr200,000 ± 15,000) while the second peak had aMrof 130,000 ± 10,000. Reconstitution experiments revealed that only the tetrameric form of the apoenzyme could be converted into an active holoenzyme while the dimeric form could not be reconstituted into an active enzyme. These results demonstrate that PLP plays an important role in maintaining the structural integrity of the enzyme by preventing the dissociation of the enzyme into subunits, in addition to its function in catalysis.