INTERACTION OF SHEEP LIVER APO-SERINE HYDROXYMETHYLTRANSFERASE WITH PYRIDOXAL-5'-PHOSPHATE - A PHYSICOCHEMICAL, KINETIC, AND THERMODYNAMIC STUDY

Sheep liver serine hydroxymethyltransferase (EC 2.1.2.1) is a homotetr amer of M(r) 213,000 requiring pyridoxal-5'-phosphate (PLP) as cofacto r, Removal of PLP from the holoenzyme converted the enzyme to the apo form which, in addition to being inactive, was devoid of the character istic absorption spectrum. Upon the addition of PLP to the apoenzyme, complete activity was restored and the visible absorption spectrum wit h a maximum at 425 nm was regained. The interaction of PLP with the ap oenzyme revealed two phases of reaction with pseudo-first-order rate c onstants of 20 +/- 5 s(-1) and 12.2 +/- 2.0 x 10(-3) s(-1), respective ly. However, addition of PLP to the apoenzyme did not cause gross conf ormational 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 degrees C, respectively, and the reconstituted h oloenzyme was thermally as stable as the native holoenzyme. These resu lts suggested that there was no apparent difference in the secondary s tructure of holoenzyme, apoenzyme, and reconstituted holoenzyme, Howev er, sedimentation analysis of the apoenzyme revealed the presence of t wo peaks of S-20,S-w values of 8.7 +/- 0.5 and 5.7 +/- 0.3 S, respecti vely. A similar pattern was observed when the apoenzyme was chromatogr aphed on a calibrated Sephadex G-150 column. The first peak correspond ed to the tetrameric form (M(r) 200,000 +/- 15,000) while the second p eak had a M(r) of 130,000 +/- 10,000. Reconstitution experiments revea led that only the tetrameric form of the apoenzyme could be converted into an active holoenzyme while the dimeric form could not be reconsti tuted into an active enzyme. These results demonstrate that PLP plays an important role in maintaining the structural integrity of the enzym e by preventing the dissociation of the enzyme into subunits, in addit ion to its function in catalysis. (C) 1996 Academic Press, Inc.