Interactions of L-serine at the active site of serine hydroxymethytransferases: Induction of thermal stability.

Serine hydroxymethyltransferase (SHMT), EC 2.1.2.1, exhibits broad substrate and reaction specificity. In addition to cleaving many
3-hydroxyamino 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 of substrates, especially L-serine with the enzyme, a
comparative study of interaction of L-serine with the enzyme from sheep liver and Escherichia coli, was carried out. The heat stability of
both the enzymes was enhanced in the presence of serine, although to different extents. Thermal denaturation monitored by spectral
changes indicated an alteration in the apparent T m of sheep liver and E. coli SHMTs from 55 + I°C to 72 + 3°C at 40 mM serine and
from 67 + I°C to 72 + I°C at 20 mM serine, respectively. Using stopped 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 determined at 50 mM serine. The binding of serine monitored by intrinsic
fluorescence 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 environment of pyridoxal 5'-phosphate at the active site upon
binding of serine to both the enzymes. The formation of an external aldimine was accompanied by a change in the secondary structure of
the enzymes monitored by far UV-CD spectra. Titration microcalorimetric studies in the presence of serine (8 mM) also demonstrated a
single class of binding and the conformational changes accompanying the binding of serine to the enzyme resulted in a more compact
structure leading to increased thermal stability of the enzyme.