Denaturation by guanidine-HCl, urea, or heating was performed on the common isoforms of human apolipoprotein (apo) E (apoE2, apoE3, and apoE4) and their 22-kDa and 10-kDa fragments in order to investigate the effects of the cysteine/arginine interchanges at residues 112 and 158. Previous physical characterization of apoE3 established that apoE contains two domains, the 10-kDa carboxyl-terminal and 22-kDa amino-terminal domains, which unfold independently and exhibit large differences in stability. However, the physical properties of apoE2, apoE3, and apoE4 have not been compared before. Analysis by circular dichroism showed that the different isoforms have identical alpha-helical contents and guanidine-HCl denaturation confirmed that the two domains unfold independently in all three isoforms. However, guanidine-HCl, urea, and thermal denaturation showed differences in stability among the 22-kDa amino-terminal fragments of the apoE isoforms (apoE4 < apoE3 < apoE2). Furthermore, guanidine-HCl denaturation monitored by circular dichroism and fluorescence suggested the presence of a folding intermediate in apoE, most prominently in apoE4. Thus, these studies reveal that the major isoforms of apoE, which are associated with different pathological consequences, exhibit significant differences in stability.

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 ␣ -helices in the 4-helix bundle formed by the 22 kDa N-terminal domain of apoE reduced the reaction rate. In contrast, the more flexible interhelical connections in apoA-I and the 10 kDa C-terminal domain of apoE promoted rapid solubilization of dimyristoyl-phosphatidylcholine (DMPC) multilamellar vesicles (mLV). Full-length apoE-3 reacted at about half the rate of the C-terminal domain alone. This decrease occurred because the hinge region probably decreased the interhelical flexibility of the 10 kDa domain and because both domains are conformationally restricted when covalently linked. Furthermore, the mLV surface affinities and reaction rates of the N-terminal domain fragments of the three common apoE isoforms tended to vary inversely with the stabilities of these fragments. These results confirm the importance of apoE's structure on the kinetics of lipid interaction. They suggest that flexibility in an apolipoprotein molecule increases the time-averaged exposure of hydrophobic surface area, thereby increasing the rate of phospholipid solubilization. Apolipoprotein E (apoE) is a constituent of several plasma lipoproteins and plays a key role in the metabolism of cholesterol and triglyceride. It directs the uptake of lipoproteins through interaction with cell-surface receptors of the LDL receptor (LDLR) family (1, 2) and heparan sulfate proteoglycans (3). In addition, apoE participates in neuronal repair in the central nervous system (CNS) (4). The single polypeptide chain of apoE (299 residues, 34.2 kDa molecular mass) contains two independently folded domains that are approximated by two thrombolytic fragments (residues 1-191 and 216-299), which are involved in different functions of the protein (5). The 22 kDa N-terminal fragment contains the LDLRbinding domain (residues 136-150) and the C-terminal 10 kDa fragment represents the major lipid-binding region. The lipid binding of apoE is mediated by amphipathic ␣ -helices (6). Polymorphism in apoE arises from three allele products designated apoE-2, apoE-3, and apoE-4 and involves point mutations at residues 112 and 158. ApoE-4 contains R at both positions, whereas apoE-3 contains C/R and apoE-2 contains C/C at these sites. The C/R interchange that distinguishes apoE-3 and apoE-4 has no effect on receptor binding, whereas the C/R substitution that defines apoE-2 dramatically reduces receptor binding (5). The presence of apoE-2 is the primary defect in type III hyperlipoproteinemia. ApoE-4 is associated with higher plasma LDL concentrations than apoE-2 or apoE-3, and apoE-2 and apoE-4 alleles have higher levels

Adenylosuccinate lyase (ADL) catalyzes the breakdown of 5-aminoimida-zole-(Nsuccinylocarboxamide) 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 α-helical, homotetrameric structure; three regions of highly conserved amino acid residues; and a general acid-base catalytic mechanism with the overall β-elimination of fumarate as a product. The crystal structures of wild-type Escherichia coli ADL (ec-ADL), and mutant-substrate (H171A-ADS) and -product (H171N-AMP•FUM) complexes have been determined to 2.0, 1.85, and 2.0 Å resolution, respectively. The H171A-ADS and H171N-AMP•FUM structures provide the first detailed picture of the ADL active site, and have enabled the precise identification of substrate binding and putative catalytic residues. Contrary to previous suggestions, the ec-ADL structures implicate S295 and H171 in base and acid catalysis, respectively. Furthermore, structural alignments of ec-ADL with other superfamily members suggest for the first time a large conformational movement of the flexible C3 loop (residues 287-303) in ec-ADL upon substrate binding and catalysis, resulting in its closure over the active site. This loop movement has been observed in other superfamily enzymes, and has been proposed to be essential for catalysis. The ADL catalytic mechanism is re-examined in light of the results presented here.