Martin Ph. Verbeet

A Random-sequential Mechanism for Nitrite Binding and Active Site Reduction in Copper-containing Nitrite Reductase

The homotrimeric copper-containing nitrite reductase (NiR) contains one type-1 and one type-2 copper center per monomer. Electrons enter through the type-1 site and are shuttled to the type-2 site where nitrite is reduced to nitric oxide. To investigate the catalytic mechanism of NiR the effects of pH and nitrite on the turnover rate in the presence of three different electron donors at saturating concentrations were measured. The activity of NiR was also measured electrochemically by exploiting direct electron transfer to the enzyme immobilized on a graphite rotating disk electrode. In all cases, the steady-state kinetics fitted excellently to a random-sequential mechanism in which electron transfer from the type-1 to the type-2 site is rate-limiting. At low [NO–2] reduction of the type-2 site precedes nitrite binding, at high [NO–2] the reverse occurs. Below pH 6.5, the catalytic activity diminished at higher nitrite concentrations, in agreement with electron transfer being slower to the nitrite-bound type-2 site than to the water-bound type-2 site. Above pH 6.5, substrate activation is observed, in agreement with electron transfer to the nitrite-bound type-2 site being faster than electron transfer to the hydroxyl-bound type-2 site. To study the effect of slower electron transfer between the type-1 and type-2 site, NiR M150T was used. It has a type-1 site with a 125-mV higher midpoint potential and a 0.3-eV higher reorganization energy leading to an ∼50-fold slower intramolecular electron transfer to the type-2 site. The results confirm that NiR employs a random-sequential mechanism.

Protein adsorption at a gold electrode studied by in situ scanning tunnelling microscopy

The insitu adsorption, under physiological conditions, of azurin molecules at a gold electrode surface has been monitored at the molecular level by scanning tunnelling microscopy. The introduction of free cysteine residues into the surface of the redox protein by structurally conservative mutagenesis allows the immobilisation to be controlled in a manner in which the protein electrochemical activity is retained.

Expression of cDNA-encoded human acid α-glucosidase in milk of transgenic mice

Enzyme replacement therapy is at present the option of choice for treatment of lysosomal storage diseases. To explore the feasibility of lysosomal enzyme production in milk of transgenic animals, the human acid α-glucosidase cDNA was placed under control of the αS1-casein promoter and expressed in mice. The milk contained recombinant enzyme at a concentration up to 1.5 μg/ml. Enzyme purified from milk of transgenic mice was internalized via the mannose 6-phosphate receptor and corrected enzyme deficiency in fibroblasts from patients. We conclude that transgenically produced human acid α-glucosidase meets the criteria for therapeutic application.

Reconstitution of the type-1 active site of the H145G/A variants of nitrite reductase by ligand insertion

Variants of the copper-containing nitrite reductase (NiR) of Alcaligenes faecalis S6 were constructed by site-directed mutagenesis, by which the C-terminal histidine ligand (His145) of the Cu in the type-1 site was replaced by an alanine or a glycine. The type-1 sites in the NiR variants as isolated, are in the reduced form, but can be oxidized in the presence of external ligands, like (substituted) imidazoles and chloride. The reduction potential of the type-1 site of NiR-H145A reconstituted with imidazole amounts to 505 mV vs NHE (20 degrees C, pH 7, 10 mM imidazole), while for the native type-1 site it amounts to 260 mV. XRD data on crystals of the reduced and oxidized NiR-H145A variant show that in the reduced type-1 site the metal is 3-coordinated, but in the oxidized form takes up a ligand from the solution. With the fourth (exogenous) ligand in place the type-1 site is able to accept electrons at about the same rate as the wt NiR, but it is unable to pass the electron onto the type-2 site, leading to loss of enzymatic activity. It is argued that the uptake of an electron by the mutated type-1 site is accompanied by a loss of the exogenous ligand and a concomitant rise of the redox potential. This rise effectively traps the electron in the type-1 site.

Cloning and characterization of the bovine polymeric immunoglobulin receptor-encoding cDNA

Trans-epithelial transport of polymeric immunoglobulins (pIg) into mucosal and glandular secretions is carried out by the pIg receptor (pIgR). Therefore, expression of the pIgR gene in epithelial cells of mucosal and glandular tissues is an absolute requirement for achieving mucosal immunity. We report the cloning and characterization of the bovine pIgR cDNA. Three overlapping cDNA clones with a total length of 3608 bp yielded an open reading frame encoding a 757-amino-acid (aa) transmembrane (TM) glycoprotein. Although polymorphism was found in two separate clones, Northern blot analysis showed a single pIgR mRNA (approx. 3.8 kb) to be present in the mammary gland, liver, lung, kidney and intestine of a lactating cow. There was no detectable expression of pIgR in the spleen of the same animal. Comparison of the deduced bovine pIgR as sequence with those of rat, mouse, man and rabbit shows that this receptor is highly conserved both in aa sequence and structural organization. The degree of conservation in the TM sequence and the C-terminal cytoplasmic tail, which contains the various signals for intracellular trafficking of the receptor, is 65-73%. We also find a high degree of conservation (61-66%) in the ectoplasmic part of the receptor, known as the secretory component (SC), with an exception for that of the rabbit SC, which is much lower (47%). Among the five Ig-like domains in the SC, the N-terminal domain I, where the primary pIg-binding site is located, showed the highest (72-83%) aa sequence conservation.

Enzyme therapy for Pompe disease: from science to industrial enterprise

Abstract. Pompe disease or glycogen storage disease type II (OMIM 232300) is a metabolic myopathy with a broad clinical spectrum. Generalised muscle weakness combined with cardiomegaly presents within the first 3 months after birth, if the lysosomal α-glucosidase (AGLU) deficiency is complete. Residual enzyme activity prevents cardiac involvement and delays onset of muscle weakness. Enzyme therapy, by intravenous administration of acid AGLU, aims to supplement the missing enzyme activity. At the SHS symposium on Glycogen Storage Diseases Type I and II, in Fulda, two interim accounts were given of studies on the efficacy of enzyme therapy for Pompe disease; one with recombinant human acid AGLU produced in Chinese hamster ovary cells and the other with the same enzyme produced in the milk of transgenic rabbits. Conclusion: this review focuses on the latter study, discusses the scientific, technological and commercial aspects of the enterprise, and addresses the prospects and challenges of enzyme therapy for Pompe disease.

Effects of Dimerization on Protein Electron Transfer

In order to investigate the relationship between the rate of protein-protein electron transfer and the structure of the association complex, a dimer of the blue copper protein azurin was constructed and its electron exchange properties were determined. For this purpose, a site for covalent cross-linking was engineered by replacing the surface-exposed asparagine 42 with a cysteine. This mutation enabled the formation of disulfide-linked homo-dimers of azurin. Based on NMR line-broadening experiments, the electron self-exchange (e.s.e.) rate constant for this dimer was determined to be 4.2(+/-0.7) x 10(5)M(-1)s(-1), which is a seven-fold decrease relative to wild-type azurin. This difference is ascribed to a less accessible hydrophobic patch in the dimer. To discriminate between intramolecular electron transfer within a dimer and intermolecular electron transfer between two dimers, the e.s.e. rate constant of (Cu-Cu)-N42C dimers was compared with that of (Zn-Cu)- and (Ag-Cu)-N42C dimers. As Zn and Ag are redox inactive, the intramolecular electron transfer reaction in these latter dimers can be eliminated. The e.s.e. rate constants of the three dimers are the same and an upper limit for the intramolecular electron transfer rate of 10 s(-1) could be determined. This rate is compatible with a Cu-Cu distance of 18 A or more, which is larger than the Cu - Cu distance of 15 A observed in the wild-type crystal structure that shows two monomers that face each other with opposing hydrophobic patches. Modelling of the dimer shows that the Cu-Cu distance should be in the range of 17 A < rCu-Cu < 28 A, which is in agreement with the experimental findings. For efficient electron transfer, it appears crucial that the two molecules interact in the proper orientation. Direct cross-linking may disturb the formation of such an optimal electron transfer complex.

The Role Played by the α-Helix in the Unfolding Pathway and Stability of Azurin: Switching Between Hierarchic and Nonhierarchic Folding

The role played by the α‐helix in determining the structure, the stability and the unfolding mechanism of azurin was addressed by studying a helix‐depleted azurin variant produced by sitedirected mutagenesis. The protein structure was investigated by CD, 1D 1H NMR, fluorescence spectroscopy measurements and MD simulations, whilst EPR, UV‐visible and cyclic voltammetry experiments were carried out to investigate the geometry and the properties of the CuII site. The effects of the α‐helix depletion on the thermal stability and the unfolding pathway of the protein were determined by DSC, UV/visible and fluorescence measurements at increasing temperature. The results show that, in the absence of the α‐helix segment, the overall protein structure is maintained, and that only the Cu site is slightly modified. In contrast, the protein stability is diminished by about 60 % with respect to the wild‐type azurin. Moreover, the unfolding pathway of the mutant azurin involves the presence of detectable intermediates. In comparison with previous studies concerning other small β‐sheet cupredoxins, the results as a whole support the hypothesis that the presence of the α‐helix can switch the folding of azurin from a hierarchic to a nonhierarchic mechanism in which the highly conserved β‐sheet core provides a scaffold for cooperative folding of the wild‐type protein.

Thermodynamic analysis of the contributions of the copper ion and the disulfide bridge to azurin stability: synergism among multiple depletions

The stabilizing potential of the copper ion and the disulfide bridge in azurin has been explored with the aim of inspecting the ways in which these two factors influence one another. Specifically, whether copper and disulfide contributions to protein stability are additive has been examined. To this aim, the thermal unfolding of a copper-depleted mutant lacking the disulfide bridge between Cys3 and Cys26 (apo C3A/C26A azurin) was studied by differential scanning calorimetry. A comparison of the unfolding parameters of holo and apo C3A/C26A azurin with the apo C3A/C26A protein has shown that the effects of simultaneous copper and disulfide depletion are additive only at two temperatures: T=15 degrees C and T=67 degrees C. Within this range the presence of the copper ion and the disulfide bridge has a positive synergistic effect on azurin stability. These findings might have implications for the rational use of the stabilizing potential of copper and disulfides in copper protein engineering.

The 1.6 Å resolution crystal structure of a mutant plastocyanin bearing a 21–25 engineered disulfide bridge

Plastocyanin is an electron-transfer protein which has been largely used for biophysical studies as well as for protein-engineering experiments. A surface disulfide bridge has been engineered in poplar plastocyanin to allow protein chemisorption on gold substrates. The mutated plastocyanin crystal structure has been studied at 1.6 A resolution (R factor = 0.145, R(free) = 0.205) to characterize the effects of the engineered disulfide on the overall protein structure and on the Cu-coordination sphere in view of biophysical applications. The new orthorhombic crystal form isolated for the mutated plastocyanin displays two protein molecules per asymmetric unit.