Bhaskar, B and Prakash, V and Savithri, HS and Rao, NA (1994) Interactions of L-serine at the active site of serine hydroxymethyltransferases: induction of thermal stability. In: Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1209 (1). pp. 40-50.
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Serine hydroxymethyltransferase (SHMT), EC 126.96.36.199, 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, 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 degrees C to 72 +/- 1 degrees 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.
|Item Type:||Journal Article|
|Additional Information:||Copyright of this article belongs to Elsevier science.|
|Department/Centre:||Division of Biological Sciences > Biochemistry|
|Date Deposited:||13 Apr 2011 05:01|
|Last Modified:||13 Apr 2011 05:01|
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