Structure and bacterial receptor activity of a human salivary proline-rich glycoprotein

Using an overlay technique, we previously showed that the Gram-negative periodontal pathogen Fusobacterium nucleatum binds to a glycoprotein of Mr 89,000 (Prakobphol, A., Murray, P., and Fischer, S.J. (1987) Anal. Biochem. 164, 5-11) in the parotid saliva of some individuals. We now show that deglycosylation of the purified glycoprotein results in loss of receptor activity. Amino acid analysis of the protein core showed predominantly proline, glycine, and glutamic acid/glutamine, a characteristic of proline-rich glycoproteins (PRG). The amino terminus contained repeating sequences of Ser-Gln-Gly-Pro-Pro-Pro-Arg-Pro-Gly-Lys-Pro-Glu-Gly-Pro-Pro-Pro- Gln-Gly that had significant compositional and sequence homology to that encoded by exon 3 of the PRB3 gene. We analyzed the PRG oligosaccharides by a combination of mass spectrometry techniques and nuclear magnetic resonance spectroscopy. Twenty-seven highly fucosylated structures were identified. The most abundant was as follows (where Fuc is fucose). (formula; see text) To understand the structural basis of F. nucleatum binding, we screened glycolipids and neoglycolipids carrying carbohydrate structures related to those of the PRG for receptor activity; components with unsubstituted terminal lactosamine residues best supported adherence. Neoglycolipids constructed from PRG oligosaccharides were also receptors. Treatment with beta-galactosidase, but not alpha-fucosidase, abolished binding, suggesting that unsubstituted lactosamine units, including the 6-antenna of the major oligosaccharide, mediate F. nucleatum adherence.     

Investigation of cis/trans ratios of peptide bonds in AVP analogues containing N-methylphenylalanine enantiomers.

The solution conformation of vasopressin analogues, modified at positions 2 and 3 with N-methylphenylalanine or its enantiomer, [D-MePhe2,MePhe3]AVP and [MePhe2,D-MePhe3]AVP, were studied by 2D NMR spectroscopy in H2O/D2O and theoretical calculations (EDMC/ANALYZE). In the case of [MePhe2,D-MePhe3]AVP, the synthesis afforded two products, A and B, which had identical molecular ions and similar retention times on HPLC. This finding was explained by racemization of Cys1, which gave an additional analogue, [D-Cys1,MePhe2,D-MePhe3]AVP (B). The possibility is not excluded of racemization of Cys1 in the remaining analogues of this series. However, only in the case of [MePhe2,D-MePhe3]AVP was this process so distinct that two strong peaks in the HPLC chromatogram were observed. The NMR spectra of all the analogues showed several distinct sets of residual proton resonances. This suggests that the peptides adopt more than two groups of conformations in H2O/D2O. This fact is due to cis/trans isomerization. Two more populated isomers arise from the cis/trans isomerization across the 2-3 peptide bond in [D-MePhe2,MePhe3]AVP and [MePhe2,D-MePhe3]AVP (A) and across the 1-2 peptide bond in [D-Cys1,MePhe2,D-MePhe3]AVP (B).     

N.m.r. analyses of the histidine microenvironments in a human salivary proline-rich glycoprotein

The pKa's of the three histidine residues in a proline-rich glycoprotein from human parotid saliva (PRG) were determined by 360 MHz proton n.m.r. spectroscopy. The addition of calcium (0.64 mM) caused drops in the pKa's of all three histidines by approximately 0.25 units. When imidazole and cyclo)L-histidine-L-proline) were used as model compounds, corresponding concentrations of calcium had no effect on their pKa's. Also, the model compounds gave absolute pKa values in good agreement with similar chemical species reported in the literature. Exchange lifetime data and previously reported hydrogen----deuterium exchange experiments suggest that the PRG histidine N tau H protons are not involved in hydrogen-bonds. Collectively, these data imply that changes in PRG conformation occur upon the addition of calcium.     

Coupling between trans/cis proline isomerization and protein stability in staphylococcal nuclease.

The nucleases A produced by two strains of Staphylococcus aureus, which have different stabilities, differ only in the identity of the single amino acid at residue 124. The nuclease from the Foggi strain of S. aureus (by convention nuclease WT), which contains His124, is 1.9 kcal.mol-1 less stable (at pH 5.5 and 20 degrees C) than the nuclease from the V8 strain (by convention nuclease H124L), which contains Leu124. In addition, the population of the trans conformer at the Lys116-Pro117 peptide bond, as observed by NMR spectroscopy, is different for the two variants: about 15% for nuclease WT and 9% for nuclease H124L. In order to improve our understanding of the origin of these differences, we compared the properties of WT and H124L with those of the H124A and H124I variants. We discovered a correlation between effects of different residues at this position on protein stability and on stabilization of the cis configuration of the Lys116-Pro117 peptide bond. In terms of free energy, approximately 17% of the increase in protein stability manifests itself as stabilization of the cis configuration at Lys116-Pro117. This result implies that the differences in stability arise mainly from structural differences between the cis configurational isomers at Pro117 of the different variants at residue 124. We solved the X-ray structure of the cis form of the most stable variant, H124L, and compared it with the published high-resolution X-ray structure of the cis form of the most stable variant, WT (Hynes TR, Fox RO, 1991, Proteins Struct Funct Genet 10:92-105). The two structures are identical within experimental error, except for the side chain at residue 124, which is exposed in the models of both variants. Thus, the increased stability and changes in the trans/cis equilibrium of the Lys116-Pro117 peptide bond observed in H124L relative to WT are due to subtle structural changes that are not observed by current structure determination technique. Residue 124 is located in a helix. However, the stability changes are too large and follow the wrong order of stability to be explained simply by differences in helical propensity. A second site of conformational heterogeneity in native nuclease is found at the His46-Pro47 peptide bond, which is approximately 80% trans in both WT and H124L. Because proline to glycine substitutions at either residue 47 or 117 remove the structural heterogeneity at that position and increase protein stability, we determined the X-ray structures of H124L + P117G and H124L + P47G + P117G and the kinetic parameters of H124L, H124L + P47G, H124L + P117G, and H124L + P47G + P117G. The individual P117G and P47G mutations cause decreases in nuclease activity, with kcat affected more than Km, and their effects are additive. The P117G mutation in nuclease H124L leads to the same local conformational rearrangement described for the P117G mutant of WT (Hynes TR, Hodel A, Fox RO, 1994, Biochemistry 33:5021-5030). In both P117G mutants, the loop formed by residues 112-117 is located closer to the adjacent loop formed by residues 77-85, and residues 115-118 adopt a type I' beta-turn conformation with the Lys116-Gly117 peptide bond in the trans configuration, as compared with the parent protein in which these residues have a typeVIa beta-turn conformation with the Lys116-Pro117 peptide bond in the cis configuration. Addition of the P47G mutation appears not to cause any additional structural changes. However, the electron density for part of the loop containing this peptide bond was not strong enough to be interpreted.     

N.m.r. and computer-simulated conformational analyses of a nonapeptide found in a human salivary proline-rich glycoprotein

The amino acid sequence G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-G(7)-K(8)-P(9) occurs twice in the proline-rich glycoprotein (PRG) found in human parotid saliva. As part of our efforts to elucidate the structure-function relationships of PRG, this nonapeptide sequence (PRG9) was synthesized for the purpose of conformational analyses by high-resolution proton n.m.r. spectroscopy and computer-modeling. The empirical n.m.r. spectrum differed from the simulated spectrum in that the overall chemical shift locations were displaced from their random coil positions and the five proline residues had non-degenerate C alpha H alpha protons. Other n.m.r. data indicated that no intramolecular hydrogen-bonding was present in the PRG. In conjunction with X-ray crystallographic data on a triproline-containing model compound (Kartha, g., Ashida, T. & Kakudo, M. (1974) Acta Cryst. B30, 1861-1866), four energy-minimized PRG9 structures were obtained. Two of the structures were energetically unfavorable, while the other two conformations were reasonable. The two most likely structures gave all prolines an S-type ring pucker, the P(2)-P(3)-P(4) sequence as a poly-L-proline II helix, the H(5) phi = -90.3 degrees, P(6) and P(9) with trans peptide bond orientation, G(7) in an extended state, and the K(8) phi = -93.2 degrees or -146.8 degrees for structures #1 and #2, respectively.     

Conformational study of Ac-Xaa-Pro-NHMe dipeptides: proline puckering and trans/cis imide bond.

The conformational study on 20 Ac-Xaa-Pro-NHMe dipeptides has been carried out using an empirical potential function ECEPP/3 in order to investigate the factors responsible for the preference of proline puckering of the peptides with the trans or cis imide bond preceding the proline. The general conformational preference for down- and up-puckered dipeptides is calculated as trans-down > trans-up > cis-down > cis-up, which is reasonably in accord with that estimated by analyzing X-ray structures of proteins and the result for the single proline residue. The overestimated occurrence of trans-down conformations of proline seems to be caused by excluding long-range interactions that short dipeptides cannot have. The average computed occurrence of dipeptides with cis imide bonds is about 3%, somewhat lower than the value calculated for Ac-Pro-NHMe, which is close to experimental estimates obtained from X-ray structures of proteins. In particular, the interaction of the aromatic side chain of Xaa residue with the proline ring appears not to be strong enough to stabilize the stacked conformations of small dipeptides with cis imide bonds. The propensity to adopt trans or cis imide bond and to form secondary structures of Xaa-Pro sequences is discussed and compared with results obtained from X-ray structures of proteins.     

A peptide model for proline isomerism in the unfolded state of staphylococcal nuclease.

Nuclear magnetic resonance spectroscopy has been used to investigate a synthetic peptide (YVYKPNNTHE) corresponding to residues 113 to 122 of staphylococcal nuclease. In the major folded state of the protein this region forms a type VIa beta-turn containing a cis Lys116-Pro117 peptide bond. There is, however, no evidence for any significant population of such a turn in the peptide in aqueous solution and the X-Pro bond is predominantly in the trans configuration. The peptide exhibits several well-resolved minor resonances due to the presence of a small fraction (4 +/- 2%) of the cis-proline isomer. The ratio of cis to trans isomer populations was found to be independent of temperature between 5 degrees C and 70 degrees C, indicating that delta H for the isomerism is close to zero. Using magnetization transfer techniques the rate of trans to cis interconversion was found to be 0.025(+/- 0.013) s-1 at 50 degrees C. The thermodynamics and kinetics of isomerism in the peptide are very similar to those estimated for the Lys116-Pro117 peptide bond in unfolded nuclease, suggesting that the cis-trans equilibrium in the unfolded protein is largely determined by the residues adjacent to Pro117 in the sequence. These results are consistent with previous suggestions that the cis-proline bond is stabilized late in the folding process and that the predominance of the cis form in folded nuclease is due to stabilizing interactions within the protein that give rise to a favorable enthalpy term.     

Structural mechanism governing cis and trans isomeric states and an intramolecular switch for cis/trans isomerization of a non-proline peptide bond observed in crystal structures of scorpion toxins

Non-proline cis peptide bonds have been observed in numerous protein crystal structures even though the energetic barrier to this conformation is significant and no non-prolyl-cis/trans-isomerase has been identified to date. While some external factors, such as metal binding or co-factor interaction, have been identified that appear to induce cis/trans isomerization of non-proline peptide bonds, the intrinsic structural basis for their existence and the mechanism governing cis/trans isomerization in proteins remains poorly understood. Here, we report the crystal structure of a newly isolated neurotoxin, the scorpion alpha-like toxin Buthus martensii Karsch (BmK) M7, at 1.4A resolution. BmK M7 crystallizes as a dimer in which the identical non-proline peptide bond between residues 9 and 10 exists either in the cis conformation or as a mixture of cis and trans conformations in either monomer. We also determined the crystal structures of several mutants of BmK M1, a representative scorpion alpha-like toxin that contains an identical non-proline cis peptide bond as that observed in BmK M7, in which residues within or neighboring the cis peptide bond were altered. Substitution of an aspartic acid residue for lysine at residue 8 in the BmK M1 (K8D) mutant converted the cis form of the non-proline peptide bond 9-10 into the trans form, revealing an intramolecular switch for cis-to-trans isomerization. Cis/trans interconversion of the switch residue at position 8 appears to be sequence-dependent as the peptide bond between residues 9 and 10 retains its wild-type cis conformation in the BmK M1 (K8Q) mutant structure. The structural interconversion of the isomeric states of the BmK M1 non-proline cis peptide bond may relate to the conversion of the scorpion alpha-toxins subgroups.     

Phosphopeptides derived from human salivary acidic proline-rich proteins. Biological activities and concentration in saliva.

Human saliva contains a large number of phosphopeptides derived by cleavage of acidic proline-rich proteins (APRPs). These peptides were purified by column chromatography and they constituted 0.5% of APRPs in parotid saliva, but 11% of APRPs in saliva expectorated from the mouth (whole saliva), indicating that there is considerable cleavage of APRPs after secretion from the gland. Similarly to APRP, the phosphopeptides bind Ca2+, but they accounted for only 4% of protein-bound Ca2+ in whole saliva. APRPs as well as the phosphopeptides inhibited formation of hydroxyapatite, but, whereas 19-20 micrograms of APRP was needed for 50% inhibition, only 0.7-3.3 micrograms of purified peptides was needed for the same degree of activity, and the phosphopeptides accounted for 18% of total inhibitory activity in whole saliva. All phosphopeptides adsorbed on hydroxyapatite in vitro, and adsorption of phosphopeptides on tooth surfaces in vivo could also be demonstrated, indicating that they would be able to inhibit unwanted mineral formation on the tooth surface in vivo. Degradation of APRPs after secretion therefore does not lead to a loss of their biological activities.     

NMR conformational analysis of cis and trans proline isomers in the neutrophil chemoattractant, N-acetyl-proline-glycine-proline

Alkaline hydrolysis of corneal proteins in the alkali-injured eye releases N-acetyl-proline-glycine-proline (Ac-Pro-Gly-Pro-OH) among other peptides. It has been shown that this tripeptide is a neutrophil chemoattractant. Existing data suggest that the release of this peptide is the catalytic event for early neutrophil invasion of the cornea leading to corneal ulcers. In order to design inhibitors of this tripeptide chemoattractant that would block neutrophil invasion and diminish corneal ulcers, we studied the solution properties of this tripeptide by NMR spectroscopy and compared this peptide to Ac-Pro-Gly-OH (a weaker chemoattractant), and to Ac-Pro-OH (inactive). The NMR data were consistent with Ac-Pro-Gly-Pro-OH existing in solution as a mixture of four isomers with different cis and trans conformations about the two X-proline amide bonds. The isomer with two trans conformations (trans-trans) was the most dominant (41%) in aqueous solution. This was followed by the isomers with mixed cis and trans conformations (trans-cis, 26% and cis-trans, 20%). The isomer with two cis conformations (cis-cis) was the least favored (13%). The populations of these isomers were investigated in DMSO and they were similar to those reported in aqueous solutions except that the ordering of the trans-cis and cis-trans isomers were reversed. NMR NH temperature coefficients and nuclear Overhauser effect (NOE) measurements as well as CD spectroscopy were used to demonstrate that the four isomers exist primarily in an extended conformation with little hydrogen bonding. The available (NOE) information was used with molecular dynamics calculations to construct a dominant solution conformation for each isomer of the tripeptide. This information will serve as a model for the design of peptide and nonpeptide inhibitors of the chemoattractant.     

Structural and functional characterization of the porcine proline-rich antifungal peptide SP-B isolated from salivary gland granules.

A 1905-Da cationic proline-rich peptide, named SP-B, was recently isolated by our group as the main component of salivary gland granules, and its primary sequence fully characterized by means of automated Edman sequencing and LC-MS/MS tools. In the present study SP-B is shown to possess antifungal activity when challenged with strains of Cryptococcus neoformans, Candida albicans and Aspergillus fumigatus, while only negligible antibacterial activity was detected. Furthermore, SP-B was found to be non-cytotoxic when tested on fibroblast cell lines. To obtain information regarding its structure affinity, capillary electrophoresis (CE), circular dichroism (CD) and attenuated total reflection (ATR)-FT/IR experiments were performed. CE revealed a pH dependence of the hydrodynamic radial dimensions both in aqueous and 2,2,2-trifluoroethanol solutions. CD and ATR-FT/IR measurements confirmed the structure-pH relationship, revealing a secondary structure composed of mixed proportions of polyproline-II, unordered and turn motifs, the last being more evident in the zwitterionic form of the peptide. From these findings SP-B peptide could be classified as a new member of the proline-rich antimicrobial peptide family.     

Circular dichroism and fluorescence spectroscopic analyses of a proline-rich glycoprotein from human parotid saliva

A proline-rich glycoprotein (PRG) was isolated from human parotid saliva and examined by circular dichroism and fluorescence spectroscopy. Addition of guanidine hydrochloride to PRG labeled with an extrinsic dansyl probe had no effect on the fluorescence spectra's 511 nm lambda-max location. Thermodynamic calculations supported the contention that PRG has no significant tertiary structure. Circular dichroism results for PRG were simulated by computer and a secondary structure composed of 70% random coil and 30% beta-form conformation was predicted. Circular dichroism of PRG failed to detect either poly-L-proline type I or II structures. Deglycosylation of PRG had no measurable effect on the circular dichroism spectrum, indicating that the carbohydrate side chains had little influence on PRG secondary structure. Based upon mathematical calculations, beta-turns were predicted around three glycosylated Asn residues of PRG. These collective data suggest that PRG is composed of a disordered polypeptide chain with at least three of the N-linked Asn residues participating in some type of beta-turn.     

Roles of a conserved proline in the internal fusion peptide of Ebola glycoprotein

The structural determinants underlying the functionality of viral internal fusion peptides (IFPs) are not well understood. We have compared EBOwt (GAAIGLAWIPYFGPAAE), representing the IFP of the Ebola fusion protein GP, and EBOwt (GAAIGLAWIPYFGRAAE) derived from a non-functional mutant with conserved Pro537 substituted by Arg. P537R substitution did not abrogate peptide-membrane association, but interfered with the ability to induce bilayer destabilization. Structural determinations suggest that Pro537 is required to preserve a membrane-perturbing local conformation in apolar environments.     

Conformational differences between cis and trans proline isomers of a peptide antigen representing the receptor binding domain of Pseudomonas aeruginosa as studied by 1H-nmr

A 17-residue disulfide-bridged peptide (PAK 128–144) corresponding to the C-terminus of Pseudomonas aeruginosa pilin strain K has been studied by one- and two-dimensional nmr techniques. This synthetic immunogen has been found to exist as two distinct conformations in solution, which have been demonstrated to arise as a result of the isomerization of the I¹³⁸-P¹³⁹ amide bond. The two isomers occur in the ratio of 3 : 1 trans to cis at 5°C. Sequential assignments for both forms have been accomplished through the use of nuclear Overhauser enhancement spectroscopy (NOESY) spectra and most side-chain resonances have been assigned using a combination of correlated spectroscopy, total correlated spectroscopy, and NOESY spectra. The presence of the cis isomer, which is considerably more predominant in the oxidized peptide, was confirmed by the observation of the characteristic NOEs between P¹³⁹ and the preceding residue. Further corroboration was given by the disappearance of the cis resonances in the spectrum of the P139A analogue of PAK 128–144. From observation of the differences in the chemical shifts and amide proton temperature coefficients of the two isomers, it is apparent that the two forms differ markedly in their solution conformation. The biological implications of the isomerization are discussed. © 1994 John Wiley & Sons, Inc.     

Induction of proline-rich glycoprotein synthesis in mouse salivary glands by isoproterenol and by tannins

Glycoproteins which contain about 45 mol% proline were dramatically induced in mouse parotid and submandibular glands by isoproterenol treatment, but these unusual proteins were not detected in control animals. These acid-soluble substances were obtained by extracting tissues with 10% trichloroacetic acid, as reported previously for isolating proline-rich proteins from rat submandibular glands (Mehansho, H., and Carlson, D.M. (1983) J. Biol. Chem. 258, 6616-6620). Three major proline-rich glycoproteins were induced in parotid glands with apparent molecular weights of 66,000 (GP-66p), 45,000 (GP-45p), and 27,000 (GP-27p), whereas only one such protein was expressed by the submandibular glands (66,000 (GP-66sm]. Both GP-66p and GP-66sm contained about 19% carbohydrate with the following molar ratios, respectively; GalNAc, 1.0, 1.0; Gal, 1.6, 2.3; GlcNAc, 0.8, 1.1; sialic acid, 0.9, 1.9. The peptide chains of GP-66p and GP-66sm appear to be identical by amino acid compositions, glycopeptide analysis, and preliminary amino acid sequencing data. Northern blot analysis of RNAs from parotid glands of normal and isoproterenol-treated rats, probed with a 32P-labeled proline-rich protein cDNA, confirmed that control animals were devoid of mRNAs encoding these proteins and that isoproterenol treatment dramatically induced expression of these genes. Feeding sorghum high in tannins caused changes in the parotid glands similar to those observed upon isoproterenol treatment, as noted earlier with rats (Mehansho, H., Hagerman, A., Clements, S., Butler, L., Rogler, J., and Carlson, D.M. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 3948-3952). These glycoproteins have high affinities for tannins as demonstrated by competitive binding curves.     

Alleles at the PRB3 locus coding for a disulfide-bonded human salivary proline-rich glycoprotein (Gl 8) and a null in an Ashkenazi Jew.

From electrophoretic analysis, we identified in the saliva of an Ashkenazi Jew a disulfide-bonded major glycoprotein variant (Gl 8) that is a product of the proline-rich protein (PRP) locus PRB3. A previous study of this variant protein misidentified it as Pa 2 and as a product of a different PRP locus. The other PRB3 allele in this individual is an apparent null. To identify the mutations, we sequenced the tandemly repetitious exon 3 (the major protein-coding portions) of both alleles. A CGT----TGT (Arg----Cys) mutation was found in one allele (PRB3Scys), which accounts for the disulfide-bonded and peroxidase-modifying properties of Gl 8. A single nucleotide insertion was found in the other allele (PRB3Mnull) that leads to a frameshift with a premature termination codon that causes an apparent lack of gene expression. Null alleles are frequent at PRP loci coding for basic and glycosylated PRPs, and the mechanism described might explain other null phenotypes among PRPs. From nucleotide comparisons, a model of intragenic unequal crossing-over is proposed to explain, in part, the generation of the PRB3Mnull allele. The Gl 8 protein variant is found in Ashkenazi Jews (gene frequency around .008) but not in the general white, black, or Japanese populations. It is interesting that products of different PRP genes, Gl 8 from PRB3 and Pa 1 from PRH1, are both disulfide bonded and probably modify salivary peroxidase (part of an important intraoral antibacterial system) through formation of disulfide-bonded heterodimers.     

Isomer-specific proteolysis of model substrates: influence that the location of the proline residue exerts on cis/trans specificity

In an effort to further develop the technique of isomer-specific proteolysis, a number of proline-containing substrates were subjected to hydrolysis in the presence of chymotrypsin, trypsin, or prolidase. The objective was to determine whether direct hydrolysis of the cis form of the substrate could occur and, if so, the extent to which it is slower than the hydrolysis of the equivalent trans form. It is shown that for both peptide and amide substrates, which contain proline at the P2 position, the cis form can be hydrolyzed directly by either chymotrypsin or trypsin, in contrast to earlier suggestions in the literature. For similar amide substrates, it was found that chymotrypsin has a lower catalytic efficiency for the cis form, relative to the trans form, by a factor of 20 000 while, for trypsin and its substrate, the cis form was cleaved about 2000 times less efficiently. Results for a trypsin substrate with proline at the P2' position, rather than the P2 position, were quite different however, since there was no indication that the cis form could be directly cleaved even at the highest enzyme concentration. There was also no indication that prolidase could cleave the dipeptide Phe-Pro when the active bond itself is in the cis form. These collective results suggest that the ability of proteases to cleave a substrate with a cis peptide bond depends strongly on the location of the cis bond relative to the active bond that is being cleaved.     

Glycosylated proline-rich peptide of human parotid saliva. Circular dichroism study.

The direct monitoring of sugars such as lactose, maltose, saccharose is not only useful at the applied point of view but also at the fundamental point of view for studying enzymology, especially in microbiology and fermentation. Benzyme systems were extensively used in solution for analytical applications in industry and medicine. The progress in the field of immobilization of bienzyme systems [1-3], especially within membranes [4-5], makes possible the production of new analytical devices. From the studies dealing with concentration profiles in artificial enzyme membranes [14], evidence was obtained for a well defined relationship between the local concentration of a metabolite and concentration of the first substrate in the bulk solution. In the described systems a substrate is transformed into glucose within a membrane, the glucose is then transformed in gluconic acid with a local oxygen consumption. The local pO2 level is linked to the glucose oxidase velocity, which is only linked to the glucose production, that is to say to the concentration of the first substrate. The enzyme electrode is based on the transformation of kinetic phenomena (reaction rates) into absolute values (local concentrations) through the diffusion-reaction coupling process. The manufacture of magnetic enzyme electrodes [6] allows convenient use of the active sensors. The pO2 electrode has some adventages, namely the specificity based on the selectivity of the gas permeable membrane and the linear relationship between the oxygen and the output of the electrode. pCO2, pH, ion electrodes give a logarithmic response as a function of the concentration. The grafting of a multienzyme system on a sensor allows a study of sequential systems in a defined context with a measurement of the local concentration of the metabolites. The tool is useful for both kinetics [4] and regulation studies [5].