<i>Cis‐trans</i> isomerization is rate‐determining in the reactivation of denatured human carbonic anhydrase II as evidenced by proline isomerase

Fransson, Cecilia; Freskgård, Per‐Ola; Herbertsson, Helena; Johansson, Anna; Jonasson, Per; Mårtensson, Lars-Göran; Svensson, Magdalena; Jonsson, Bengt‐Harald; Carlsson, Uno

doi:10.1016/0014-5793(92)80410-i

Cited by 59 publications

(48 citation statements)

References 27 publications

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“…Recent studies of mutants of human carbonic anhydrase I1 confirmed that cis-trans isomerization of proline is a rate- determining factor in the reactivation of denatured protein (Fransson et al, 1992). Importantly, the more energetically favored trans conformation is adopted in the open form, influencing the equilibrium in that direction.…”

Section: Discussionmentioning

confidence: 99%

Two conformational states of Candida rugosa lipase

et al. 1994

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The structure of Candida rugosa lipase in a new crystal form has been determined and refined at 2.1 A resolution. The lipase molecule was found in an inactive conformation, with the active site shielded from the solvent by a part of the polypeptide chain-the flap. Comparison of this structure with the previously determined "open" form of this lipase, in which the active site is accessible to the solvent and presumably the substrate, shows that the transition between these 2 states requires only movement of the flap. The backbone NH groups forming the putative oxyanion hole d o not change position during this rearrangement, indicating that this feature is preformed in the inactive state. The 2 lipase conformations probably correspond to states at opposite ends of the pathway of interfacial activation. Quantitative analysis indicates a large increase of the hydrophobic surface in the vicinity of the active site. The flap undergoes a flexible rearrangement during which some of its secondary structure refolds. The interactions of the flap with the rest of the protein change from mostly hydrophobic in the inactive form to largely hydrophilic in the "open" conformation. Although the flap movement cannot be described as a rigid body motion, it has very definite hinge points at Glu 66 and at Pro 92. The rearrangement is accompanied by a cis-trans isomerization of this proline, which likely increases the energy required for the transition between the 2 states, and may play a role in the stabilization of the active conformation at the water/lipid interface. Carbohydrate attached at Asn 351 also provides stabilization for the open conformation of the flap. Keywords: crystallography; interfacial activation; lipasesLipases of known 3-dimensional structure show significant similarities in their topologies and conform in full or in part to the a/fl hydrolase fold ( Ollis et al., 1992). The catalytic machinery of lipases consists of a serine protease-like triad, Ser-His-Asp/Glu, and their hydrolysis of ester bonds of triacylglycerols is thought to involve an enzymatic mechanism similar to that of the serine proteases (Chapus et al., 1988). The activity of lipases is dramatically enhanced by the presence of a lipid/water interface (Desnuelle, 1972), and it is now well accepted that the phenomenon of interfacial activation involves a conformational change in the enzyme. This has been documented for 2 lipases, one from Rhizomucor miehei (Brzozowski et ai., 199i) and another from human pancreas (van Tilbeurgh et al., 1993), in which binding of an inhibitor or substrate analog causes a conformational rearrangement of 1 or more loops near the active site, exposing the serine nucleophile to the solvent and creating also an oxyanion hole in the process.A similar rearrangement was also proposed for larger lipases from Geotrichum candidurn (GCL;Schrag & Cygler, 1993) Candida rugosa (CRL; Grochubki et ai., 1993). These 2 enzymes show -40% amino acid sequence identity, and their overall 3D structures are very similar (Grochulski ...

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Section: Discussionmentioning

confidence: 99%

Two conformational states of Candida rugosa lipase

et al. 1994

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“…Consistent with this hypothesis, Fransson et al observed that incorporating PPIase in the refolding reaction mixture of GuHCl-denatured HCA II decreased the half-time of folding from 9 to 4 min and removed the effect of time delay in refolding. 667 Further, the addition of Cyclosporin A, a specific inhibitor of PPIase, 742 completely abolished the observed PPIasemediated acceleration of folding. If PPIase acted efficiently on all peptidyl-Pro bonds, the expected half-time of refolding would decrease to 1 min.…”

Section: Isomerization Of Pro Residuesmentioning

confidence: 95%

“…Proline isomerization is the rate-limiting step in the folding of CA. 667,[680][681][682][683][684] 4. After denaturation with urea, guanidinium hydrochloride (GuHCl), heat, or SDS, CA renatures spontaneously after removal of the denaturant.…”

Section: Ca As a Model Protein For Studying The Denaturation And Renamentioning

confidence: 99%

Carbonic Anhydrase as a Model for Biophysical and Physical-Organic Studies of Proteins and Protein−Ligand Binding

et al. 2008

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I. Introduction to Carbonic Anhydrase (CA) and to the Review 948 1. Introduction: Overview of CA as a Model 948 1.1. Value of Models 950 1.2. Objectives and Scope of the Review 950 2. Overview of Enzymatic Activity 950 3. Medical Relevance 951 II. Structure and Structure−Function Relationships of CA 953 4. Global and Active-Site Structure 953 4.1. Structure of Isoforms 953 4.2. Isolation and Purification 954 4.3. Crystallization 954 4.4. Structures Determined by X-ray Crystallography and NMR 955 4.4.1. Structures Determined by X-ray Crystallography 955 4.4.2. Structure Determined by NMR 955 4.5. Global Structural Features 963 4.6. Structure of the Binding Cavity 964 4.7. Zn II -Bound Water 965 5. Metalloenzyme Variants 966 6. Structure−Function Relationships in the Catalytic Active Site of CA 968 6.1. Effects of Ligands Directly Bound to Zn II 968 6.2. Effects of Indirect Ligands 969 7. Physical-Organic Models of the Active Site of CA 969 III. Using CA as a Model to Study Protein−Ligand Binding 970 8. Assays for Measuring Thermodynamic and Kinetic Parameters for Binding of Substrates and Inhibitors 970 8.1. Overview 970 8.

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“…The measurements of the regain in CO 2 hydration activity during refolding were performed as described previously. 14 The concentration of HCA II pwt during refolding was 0.83 lM and the concentration of Cyp18 and Cyp18 R55A was 8.3 lM, and when applicable CsA was added to a concentration of 25 lM. All solutions were buffered with 0.1M Tris-H 2 SO 4 , pH 7.5.…”

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confidence: 99%

A nonessential role for Arg 55 in cyclophilin18 for catalysis of proline isomerization during protein folding

Moparthi¹,

Hammarström²,

Carlsson³

2009

Protein Science

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The protein folding process is often in vitro rate-limited by slow cis-trans proline isomerization steps. Importantly, the rate of this process in vivo is accelerated by prolyl isomerases (PPIases). The archetypal PPIase is the human cyclophilin 18 (Cyp18 or CypA), and Arg 55 has been demonstrated to play a crucial role when studying short peptide substrates in the catalytic action of Cyp18 by stabilizing the transition state of isomerization. However, in this study we show that a R55A mutant of Cyp18 is as efficient as the wild type to accelerate the refolding reaction of human carbonic anhydrase II (HCA II). Thus, it is evident that the active-site located Arg 55 is not required for catalysis of the rate-limiting prolyl cis-trans isomerization steps during the folding of a protein substrate as HCA II. Nevertheless, catalysis of cis-trans proline isomerization in HCA II occurs in the active-site of Cyp18, since binding of the inhibitor cyclosporin A abolishes rate acceleration of the refolding reaction. Obviously, the catalytic mechanisms of Cyp18 can differ when acting upon a simple model peptide, four residues long, with easily accessible Pro residues compared with a large protein molecule undergoing folding with partly or completely buried Pro residues. In the latter case, the isomerization kinetics are significantly slower and simpler mechanistic factors such as desolvation and/or strain might operate during folding-assisted catalysis, since binding to the hydrophobic active site is still a prerequisite for catalysis.

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Cis‐trans isomerization is rate‐determining in the reactivation of denatured human carbonic anhydrase II as evidenced by proline isomerase

Cited by 59 publications

References 27 publications

Two conformational states of Candida rugosa lipase

Two conformational states of Candida rugosa lipase

Carbonic Anhydrase as a Model for Biophysical and Physical-Organic Studies of Proteins and Protein−Ligand Binding

A nonessential role for Arg 55 in cyclophilin18 for catalysis of proline isomerization during protein folding

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