Effect of Asp69 and Arg310 on the pK of His68, a key catalytic residue of adenylosuccinate lyase.

Adenylosuccinate lyase (ASL) of Bacillus subtilis contains three conserved histidines, His(68), His(89), and His(141), identified by affinity labeling and site-directed mutagenesis as critical to the intersubunit catalytic site. The pH-V(max) profile for wild-type ASL is bell-shaped (pK (1) = 6.74 and pK (2) = 8.

Substrate and product complexes of Escherichia coli adenylosuccinate lyase provide new insights into the enzymatic mechanism.

Adenylosuccinate lyase (ADL) catalyzes the breakdown of 5-aminoimidazole- (N-succinylocarboxamide) ribotide (SAICAR) to 5-aminoimidazole-4-carboxamide ribotide (AICAR) and fumarate, and of adenylosuccinate (ADS) to adenosine monophosphate (AMP) and fumarate in the de novo purine biosynthetic pathway. ADL belongs to the argininosuccinate lyase (ASL)/fumarase C superfamily of enzymes. Members of this family share several common features including: a mainly alpha-helical, homotetrameric structure; three regions of highly conserved amino acid residues; and a general acid-base catalytic mechanism with the overall beta-elimination of fumarate as a product.

Important roles of hydroxylic amino acid residues in the function of Bacillus subtilis adenylosuccinate lyase.

Thr(93), Ser(94), Thr(140), and Ser(306) are conserved in all adenylosuccinate lyases (ASL) and are close to other amino acids previously identified by mutagenesis as being in the active site. To test their involvement in the enzyme's function, each of these amino acids was replaced by alanine. All the mutants exhibit circular dichroism spectra which are similar to that of wild-type enzyme, indicating there is no appreciable change in secondary structure.

Gln212, Asn270, and Arg301 are critical for catalysis by adenylosuccinate lyase from Bacillus subtilis.

In adenylosuccinate lyase from Bacillus subtilis, Gln(212), Asn(270), and Arg(301) are conserved and located close to the succinyl moiety of docked adenylosuccinate. We constructed mutant enzymes with Gln(212) replaced by Glu and Met, Asn(270) by Asp and Leu, and Arg(301) by Gln or Lys. The wild-type and mutant enzymes were expressed in Escherichia coli and purified to homogeneity.

Influence of apoE domain structure and polymorphism on the kinetics of phospholipid vesicle solubilization.

We examined the effects of apolipoprotein E (apoE) domain structure and polymorphism on the kinetics of solubilization (clearance) of dimyristoyl-phosphatidylcholine multilamellar vesicles. This second order reaction consisted of two simultaneous kinetic phases; it also exhibited saturable kinetics when the apolipoprotein concentration was increased at a constant lipid concentration. Rigid connections between alpha-helices in the 4-helix bundle formed by the 22 kDa N-terminal domain of apoE reduced the reaction rate.

Comparison of the stabilities and unfolding pathways of human apolipoprotein E isoforms by differential scanning calorimetry and circular dichroism.

Differential scanning calorimetry and circular dichroism experiments were performed to study structural differences among the common isoforms of human apolipoprotein E (apoE2, apoE3, and apoE4) and their N-terminal, 22-kDa fragments. Here, we examine thermodynamic properties that characterize the structural differences among isoforms, and also differences in their unfolding behavior. The 22-kDa fragments and their full-length counterparts were found to exhibit similar differences in thermal stability (apoE4 View Article and Find Full Text PDF