Georges Noat

Purification and characterization of a fatty acyl-ester hydrolase from post-germinated sunflower seeds

Fatty acyl-ester hydrolase was not detectable in dry sunflower seeds using various p-nitrophenyl-acyl-esters, 1,2-O-didodecyl-rac-glycero-3-glutaric acid-resorufin ester or emulsified sunflower oil as substrate. After inhibition of the seeds, acyl-ester hydrolase activity slowly developed in cotyledon extracts and was maximal after 5 days. No activity was directly measurable on oil bodies. The enzyme was purified 615-fold to apparent homogeneity, as determined by performing SDS-PAGE electrophoresis, and biochemically characterized. With p-nitrophenyl-caprylate the optimum pH was around 8.0. The purification procedure involved an acetone powder from 5-day dark-germinated seedlings, chloroform-butanol extraction and three chromatography steps. We obtained 35 micrograms of pure enzyme from 250 g of fresh cotyledons with an activity yield of around 7%. It should be possible to subsequently improve this low recovery as we gave priority here, in the first instance, to purity at the expense of the yield. The enzyme consisted of one glycosylated polypeptide chain with a molecular mass of approx. 45 kDa and, as far as we could tell, it did not seem to require metal ions to be fully active, as it was not inhibited by EDTA or o-phenanthroline and not activated by various mono or bivalent metal ions. The amino acid composition showed the presence of four cysteine and four tryptophan residues. The enzyme was partially inhibited by dithiothreitol, DTNB and PCMB. The fact that high inhibition was observed in the presence of PMSF indicates that a serine residue may possibly be involved in the catalytic mechanism. The hydrophobicity index was about 53.6% which places this enzyme in the class of the soluble proteins in good agreement with the fact that it was mainly present in the soluble part of the crude extract. Partial characterization of glycan chains, using antiglycan antibodies, showed the presence of complex Asn-linked glycans. The enzyme was active on purified sunflower glycerol derivatives. It was also able to hydrolyze monooleyl and dioleyl glycerols, as well as phosphatidylcholine, but not cholesteryl esters. Using p-nitrophenyl-acyl-esters as substrate, the highest activity was observed with middle-chain derivatives (C6 and C8). Its maximum activity was about 0.015 units mg-1 with sunflower oil. It was not activated by an organic solvent such as isooctane. This enzyme probably is involved in acyl-ester hydrolysis which follows triacylglycerol mobilization by true lipases.

Subunit interactions in enzyme transition states--antagonism between substrate binding and reaction rate.

The principles of structural kinetics as applied to polymeric enzymes have been reinvestigated in order to take account of the probable existence of subunit interactions in the enzyme transition states. On the basis of simple and plausible postulates, structural rate equations have been derived for dimeric enzymes and compared to substrate binding isotherms. It then becomes possible to understand how subunit interactions affect substrate affinity and enzyme reaction rate. There exists an antagonism between substrate binding to the enzyme and the steady state rate of product appearance. If subunit interactions increase the rate of product appearance, they decrease the fractional saturation of the enzyme by the substrate. Alternatively, if they decrease the reaction velocity they increase the fractional saturation. This seemingly paradoxical effect is the direct consequence of subunit interactions occurring in both the ground and the transition states.

Catalytic efficiency, kinetic co-operativity of oligomeric enzymes and evolution.

The catalytic performance of an enzyme, whether it is monomeric or oligomeric, depends on extra costs of energy in passing from the initial ground state to the various transition states, along the reaction co-ordinate. The improvement, during evolution, of the catalytic performance of individual subunits implies that three structural requirements are met in the course of an enzyme reaction: the unstrained enzyme subunits exist in the ground states under two conformations, one corresponding to the non-liganded state and the other to the liganded state; the inter-subunit strain is relieved in the various transition states; the subunits bound to the various transition states S not equal to, X not equal to and P not equal to have the same conformation. These structural requirements are precisely those which have been used to derive structural rate equations for polymeric enzymes. When subunits are loosely coupled, their arrangement controls the various rate constants, but not the extra costs of energy required to reach the various transition states. Moreover, one cannot expect the rate curve to display any sigmoidicity under these conditions. If subunits are tightly coupled and if the strained non-liganded and half-liganded states are destabilized with respect to the corresponding unstrained states, that is if they contain more conformational energy, the oligomeric enzyme is more catalytically efficient than the ideally isolated subunits. Moreover, if the available conformational energy of the half-liganded state is more than twice that of the non-liganded state, kinetic co-operativity is positive and the rate curve is sigmoidal. It is therefore the extent of inter-subunit strain in the half-liganded state which controls the appearance of sigmoidal kinetic behaviour. If subunits are tightly coupled but if inter-subunit strain is relieved in both the non-liganded and fully-liganded states, the half-liganded state controls both the catalytic efficiency of the enzyme and the sigmoidicity of the rate curve. Sigmoidicity and high catalytic efficiency are to be observed when this half-liganded state is destabilized relative to the corresponding unstrained state.(ABSTRACT TRUNCATED AT 400 WORDS)

Purification and properties of N-acetyl-β-d-glucosaminidase from Hevea brasiliensis latex

Abstract β-N-Acetylglucosaminidase from Hevea brasiliensis latex was isolated by anion exchange and gel filtration chromatography. The purified enzyme showed a single protein band on native electrophoresis. It is a glycoprotein with a molecular mass of 92 kDa, consisting of two 46-kDa subunits and has a glycosidic content of 16%. It shows optimal activity at pH 6.0 and at 50°C; its amino acid composition has been established. It is inhibited by several monovalent or divalent ions. The enzyme hydrolyses p- nitrophenyl -N- acetyl -β- d - glucosaminide (pNP-β- d -GlcNAc) with apparent Km and Vm values of 1.13 mM and 185 mM min−1 mg−1 of protein, respectively, at the optimum pH. p- Nitrophenyl -N- acetyl -β- d - galactosaminide (pNP-β- d -GalNAc) can also be used as a substrate but is less efficient. Glucosamine and glactosamine were competitive inhibitors with Ki values of 2.2 mM and 7.5 mM, respectively, at pH 6.0. The results of kinetic studies suggest that two ionizable groups with pK values of about 4 and 6.5 may take part in the reaction, possibly in the substrate binding. The vacuolar location of the enzyme is in agreement with the idea that the latex might play a lysosomal role in interacellular digestion processes.

Subunit coupling and kinetic co-operativity of polymeric enzymes. Amplification, attenuation and inversion effects

The principles of structural kinetics, as applied to dimeric enzymes, allow us to understand how the strength of subunit coupling controls both substrate-binding co-operativity, under equilibrium conditions, and kinetic co-operativity, under steady state conditions. When subunits are loosely coupled, positive substrate-binding co-operativity may result in either an inhibition by excess substrate or a positive kinetic co-operativity. Alternatively, negative substrate-binding co-operativity is of necessity accompanied by negative kinetic co-operativity. Whereas the extent of negative kinetic co-operativity is attenuated with respect to the corresponding substrate-binding co-operativity, the positive kinetic co-operativity is amplified with respect to that of the substrate-binding co-operativity. Strong kinetic co-operativity cannot be generated by a loose coupling of subunits. If subunit is propagated to the other, the dimeric enzyme may display apparently surprising co-operativity effects. If the strain of the active sites generated by subunit coupling is relieved in the non-liganded and fully-liganded states, both substrate-binding co-operativity and kinetic co-operativity cannot be negative. If the strain of the active sites however, is not relieved in these states, negative substrate-binding co-operativity is accompanied by either a positive or a negative co-operativity. The possible occurrence of a reversal of kinetic co-operativity, with respect to substrate-binding co-operativity, is the direct consequence of quaternary constraints in the dimeric enzyme. Moreover, tight coupling between subunits may generate a positive kinetic co-operativity which is not associated with any substrate-binding co-operativity. In other words a dimeric enzyme may well bind the substrate in a non co-operative fashion and display a positive kinetic co-operativity generated by the strain of the active sites.

Catalytic Properties and Tentative Function of a Cell Wall β-Glucosyltransferase From Soybean Cells Cultured in vitro

Summary The β-glucosidase, isolated and purified from walls of soybean cells in sterile culture, is active on the hydrolysis of hemicelluloses, but has little activity on pectic compounds. This enzyme is an exo-β-glucosidase and is devoid of any endo-β-glucosidase activity. Moreover, in addition to hydrolysis, the enzyme is able to function as a true glucosyltransferase and can transfer the glucosyl moiety of a donor to an acceptor such as gentiobiose or cellobiose. This transfer process, repeated several times, leads to the in vitro synthesis of polysaccharides with a degree of polymerization of 3, 4, 5 and even higher. The enzyme activity, determined at the outer surface of intact soybean cells, increases during cell elongation. Taken together these results suggest that, during cell extension, the enzyme transfers glucosyl moieties of hemicellulose precursors, synthesized in Golgi vesicles, to the growing end of cell wall hemicellulose chains.

pH-Regulation of acid phosphatase of plant cell walls: An example of adaptation to the intracellular milieu

Plant cell walls contain several hydrolases [l-4]. Some of them play a central role in cell extension by promoting local hydrolysis of acidic cell wall polysaccharides. Others control hydrolysis and transport of extracellular metabolites in the cell. In sycamore (Acer pseudoplutunus) cells cultured in vitro, the most abundant of these enzymes is an acid phosphatase whose activity may be detected at the outer surface of unbroken cells [5]. Owing to the polyanionic nature of primary plant cell walls, the local pH within the cell-wall matrix may be quite different from the one prevailing in the bulk phase. This difference may be as large as several pH units (61. Moreover, the Donnan potential in the cell wall matrix, generated by negative fixed charges of polygalacturonates, is tightly controlled by ionic strength of the outer bulk phase; that is, the ionic strength of the solution outside the cell. Therefore depending on the experimental conditions, the local pH in the cell wall may vary by several pH units. tured in vitro in liquid medium under sterile conditions, as in [5]. Cell disruption was done in a French press under 1000 kg/cm2. Cell wall preparations free of cytoplasmic contaminations were obtained as in [5]. About 55% of cell-wall acid phosphatase activity was solubilized by raising the ionic strength of the cell wall fragment suspension. The enzyme was purified to homogeneity from this soluble extract as in [5]. Acid phosphatase on cell wall fragments does not result from an artefact created by cell disruption because the same enzyme may be obtained by raising the ionic strength of a suspension of intact unbroken cells. This enzyme is a monomeric glycoprotein of 100 000 Mr [5].

Enzyme reactions at the surface of living cells: II. Destabilization in the membranes and conduction of signals

The dynamics of a bound-enzyme reaction is studied when the diffusion of both the substrate and the product is coupled to their electric repulsion and to enzyme reaction. Contrary to what is occurring when substrate diffusion is uncoupled with electric repulsion and enzyme reaction, no hysteresis loop of the partition coefficient exists. The electric partition coefficient monotonically declines as substrate or product concentration is increased in the reservoir. The random perturbation of a steady state may generate a localized destabilization of substrate and product concentration. This destabilization must propagate in the membrane and may be viewed as the conduction of a signal. These conduction phenomena are entirely due to electric effects. In the absence of these effects, the system is homeostatic, that is it returns back to its initial steady state after a perturbation. Obviously under these conditions conduction of signals cannot occur. Increasing the ionic strength of the external milieu tends to stabilize the system and to suppress conduction effects in the membrane.

Purification and Molecular Properties of a Cell-Wall β-Glucosyltransferase From Soybean Cells Cultured In Vitro

Summary A β-glucosidase has been isolated and purified to apparent homogeneity from soybean cell walls. The enzyme is a glycoprotein with a molecular weight close to 60 000 and a sedimentation constant equal to 4.17. The protein is made up with one polypeptide chain only.

Evolution of regulatory enzymes towards functional simplicity

Abstract The potential kinetic complexity of polymeric regulatory enzymes does not seem to be often expressed in nature. Most of these enzymes exhibit in fact a rather simple kinetic behaviour. This functional simplicity is probably the consequence of constraints between rate constants or of blocking of some reaction steps. Functional simplicity is believed to have emerged in the course of neo-Darwinian evolution as a consequence of a trend towards an improved functional efficiency. Functional efficiency may be reached, in polymeric regulatory enzymes, when either of the two sets of conditions are met. The first set of conditions implies the occurrence of the unicity of enzyme conformation in any transition state, a loose coupling between subunits and an exact balance of the driving forces exerted by the enzyme in the forward and backward directions of the catalytic step. This situation results in constraints between rate constants which allow degenerescence of the steady state rate equation. The second set of conditions involves again the unicity of enzyme conformation in any of the transition states, associated with a tight coupling of subunits, and a driving force exerted by the enzyme much strongly in the forward than in the backward direction of the catalytic step. These conditions imply blocking of some reaction steps and again degenerescence of the corresponding rate equation. The most frequent types of quaternary structure and subunit interactions, namely loose coupling between subunits, and tight coupling associated with conservation of at least one symmetry axis, have probably emerged as molecular organizations, which precisely allow both functional efficiency and simplicity to occur. Indeed these situations probably represent the term of two different evolutionary trends. Therefore enzymes that have not reached this state usually exhibit more complex kinetic behaviour. Wavy curves, “bumps” and turning points may be considered as manifestations of the ancestral character of an enzyme.