INTERACTIONS OF L-SERINE AT THE ACTIVE-SITE OF SERINE HYDROXYMETHYLTRANSFERASES - INDUCTION OF THERMAL-STABILITY

Serine hydroxymethyltransferase (SHMT), EC 2.1.2.1, exhibits broad sub strate and reaction specificity. In addition to cleaving many 3-hydrox yamino acids to glycine and an aldehyde, the enzyme also catalyzed the decarboxylation, transamination and racemization of several substrate analogues of amino acids. To elucidate the mechanism of interaction o f substrates, especially L-serine with the enzyme, a comparative study of interaction of L-serine with the enzyme from sheep liver and Esche richia coli, was carried out. The heat stability of both the enzymes w as enhanced in the presence of serine, although to different extents. Thermal denaturation monitored by spectral changes indicated an altera tion in the apparent T, of sheep liver and E. coli SHMTs from 55 +/- 1 degrees C to 72 +/- 3 degrees C at 40 mM serine and from 67 +/- 1 deg rees C to 72 +/- 1 degrees C at 20 mM serine, respectively. Using stop ped flow spectrophotometry k values of (49 +/- 5)(.)10(-3) s(-1) and ( 69 +/- 7).10(-3) s(-1) for sheep liver and E. coli enzymes were determ ined at 50 mM serine. The binding of serine monitored by intrinsic flu orescence and sedimentation velocity measurements indicated that there was no generalized change in the structure of both proteins. However, visible CD measurements indicated a change in the asymmetric environm ent of pyridoxal 5'-phosphate at the active site upon binding of serin e to both the enzymes. The formation of an external aldimine was accom panied by a change in the secondary structure of the enzymes monitored by far UV-CD spectra. Titration microcalorimetric studies in the pres ence of serine (8 mM) also demonstrated a single class of binding and the conformational changes accompanying the binding of serine to the e nzyme resulted in a more compact structure leading to increased therma l stability of the enzyme.