Abstract
Serine hydroxymethyltransferase has a conserved histidine residue (His-228) next to the lysine residue (Lys-229) which forms the internal aldimine with pyridoxal 5'-phosphate. This histidine residue is also conserved at the equivalent position in all amino acid decarboxylases and tryptophan synthase. Two mutant forms of Escherichia coli serine hydroxymethyltransferase, H228N and H228D, were constructed, expressed, and purified. The properties of the wild type and mutant enzymes were studied with substrates and substrate analogs by differential scanning calorimetry, circular dichroism, steady state kinetics, and rapid reaction kinetics. The conclusions of these studies were that His-228 plays an important role in the binding and reactivity of the hydroxymethyl group of serine in the one-carbon-binding site. The mutant enzymes utilize substrates and substrate analogs more effectively for a variety of alternate non-physiological reactions compared to the wild type enzyme. As one example, the mutant enzymes cleave L-serine to glycine and formaldehyde when tetrahydropyteroylglutamate is replaced by 5-formyltetrahydropteroylglutamate. The released formaldehyde inactivates these mutant enzymes. The loss of integrity of the one-carbon-binding site with L-serine in the two mutant forms of the enzyme may be the result of these enzymes not undergoing a conformational change to a closed form of the active site when serine forms the external aldimine complex.
Highlights
Serine hydroxymethyltransferase has a conserved acid substrate
The properties of the wild type and mutant enzymes were studied with substrates and substrate analogs by differential scanning calorimetry, circular dichroism, steady statekinetics, and rapid reaction kinetics.The conclusions of these studies were that His-228 plays an important role in the binding and reactivityof the hydroxymethyl group of serine in the one-carbon-binding site
In an attempt t o define the role of His-228in E. coli SHMT, we have investigated two mutant forms of this enzyme, H228N and H228D, and conclude that this residue plays a critical role in determining reaction specificity
Summary
T,,a,nd AH,for the denaturationof SHMT by DSC has been shown to be a sensitive methodfor comparing conformational states of rabbit liver cSHMT (Schirch et al, 1991) This is true of the values for AH, which are determined by the heat absorbed gioning from the native to the denatured state.Theproperties of E. coli wtSHMT and the mutant. Previous studies with rabbit cytosolic SHMT showed that glycine and 5-CHO-H4PteGluform a dead-end complex with the enzyme (structure ZZ, Scheme 11) that exhibits thermal denaturation properties similar to tehnezyme-serine complex (Schirch et al, 1991).
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