Comparison of hydrolytic activity in water and heptane for thirty-two commercial lipase preparations

The protein content and the rates of hydrolysis of p-nitrophenyl palmitate (pNPP) in water (soluble enzyme and emulsified substrate) and in heptane (soluble substrate and insoluble enzyme) were measured for thirty-two commercial lipase preparations. The protein content of the powders varied in a wide range as well as the activity on emulsified pNPP showing the high heterogeneity of the commercial samples. Activity in heptane also varied but to a lesser extent than that in water. There was no direct correlation between activities in water and in heptane as assayed with the same hydrolytic reaction. The ratio of activity in heptane to that in water, R(O/A) ratio, was introduced to characterize activity in organic media. Six lipases showed R(O/A) values higher than 1 demonstrating a higher activity in organic solvent than in water. A linear correlation of R(O/A) with activity in water (log plot) suggested the strong influence of diffusional limitations on activity of solid enzyme suspended in organic solvents.     

Hydrogen exchange of lysozyme powders. Hydration dependence of internal motions

The rate of exchange of the labile hydrogens of lysozyme was measured by out-exchange of tritium from the protein in solution and from powder samples of varied hydration level, for pH 2, 3, 5, 7, and 10 at 25 degrees C. The dependence of exchange of powder samples on the level of hydration was the same for all pHs. Exchange increased strongly with increased hydration until reaching a rate of exchange that is constant above 0.15 g of H2O/g of protein (120 mol of H2O/mol of protein). This hydration level corresponds to coverage of less than half the protein surface with a monolayer of water. No additional hydrogen exchange was observed for protein powders with higher water content. Considered in conjunction with other lysozyme hydration data [Rupley, J. A., Gratton, E., & Careri, G. (1983) Trends Biochem. Sci. (Pers. Ed.) 8, 18-22], this observation indicates that internal protein dynamics are not strongly coupled to surface properties. The use of powder samples offers control of water activity through regulation of water vapor pressure. The dependence of the exchange rate on water activity was about fourth order. The order was pH independent and was constant from 114 to 8 mol of hydrogen remaining unexchanged/mol of lysozyme. These results indicate that the rate-determining step for protein hydrogen exchange is similar for all backbone amides and involves few water molecules. Powder samples were hydrated either by isopiestic equilibration, with a half-time for hydration of about 1 h, or by addition of solvent to rapidly reach final hydration. Samples hydrated slowly by isopiestic equilibration exhibited more exchange than was observed for samples of the same water content that had been hydrated rapidly by solvent addition. This difference can be explained by salt and pH effects on the nearly dry protein. Such effects would be expected to contribute more strongly during the isopiestic equilibration process. Solution hydrogen exchange measurements made for comparison with the powder measurements are in good agreement with published data. Rank order was proven the same for all pHs by solution pH jump experiments. The effect of ionic strength on hydrogen exchange was examined at pH 2 and pH 5 for protein solutions containing up to 1.0 M added salt. The influence of ionic strength was similar for both pHs and was complex in that the rate increased, but not monotonically, with increased ionic strength.     

H/D isotope effects on protein hydration and interaction in solution

An approach has been suggested to study the H/D isotope effect on protein-water and protein-protein intermolecular interactions by determining the content of non-freezing water using low-temperature (1)H NMR in mixed (H2O/D2O) water solutions. Direct data are obtained on the amount of H2O adsorbed (absolute hydration) in presence of the heavy isotope (deuterium D), and isothermals of H2O/D2O fractionation at protein surface groups are presented for temperatures between -10 degrees C and -35 degrees C and solutions of varying composition. The fractionation factor, phi = [x/(1 - x)]/[x(0)/(1 - x(0))], where x and x(0) are the fractions of deuterons in hydration and bulk water, respectively, appeared to be extremely high: phi > 1 at 0.03 < x(0) < 0.10. The high values of phi indicate a decrease in apparent hydration of protein molecules. A probable reason of the effect can be an inter-protein molecular solvent-mediated interaction induced by D2O. The excess of phi over 1 appears to provide a quantitative estimate of the fraction of hydration water affected by such interaction.     

胃蛋白酶水解绿豆分离蛋白的工艺

选用胃蛋白酶对绿豆分离蛋白进行酶法水解,考察了原料预处理条件、pH、温度、底物浓度等对酶解的影响,结果表明：原料预处理最适条件为沸水浴中90℃处理20min,在37℃、pH1．8、底物质量分数7％、酶量6000U／g条件下酶解180min,水解度（DH％）为19．86％,达到了制备小肽的水解度要求。实验证明,经过水解,绿豆分离蛋白各功能特性得到很好的改善。     

A new strategy for the study of oligomeric enzymes: gamma-glutamyltransferase in reversed micelles of surfactants in organic solvents

A heterodimeric enzyme (gamma-glutamyltransferase) was studied in the reversed micellar medium of Aerosol OT (AOT) in octane. As was shown earlier, the size (radius) of inner cavity of the AOT-reversed micelles is determined by their hydration degree, i.e., [H2O]/[AOT] molar ratio, in the system. Owing to this, the dependence of hydrolytic, transpeptidation and autotranspeptidation activities of the enzyme on the hydration degree was investigated using L- and D-isomers of gamma-glutamyl(3-carboxy-4-nitro)anilide and glycylglycine as substrates. For all of the reaction types, the observed dependences are curves with three optima. The optima are found at the hydration degrees, [H2O]/[AOT] = 11, 17 and 26 when the inner cavity radii of reversed micelles are equal to the size of light (Mr 21,000) and heavy (Mr 54,000) subunits of gamma-glutamyltransferase, and to their dimer (Mr 75,000), respectively. Ultracentrifugation experiments showed that a change of the hydration degree resulted in a reversible dissociation of the enzyme to light and heavy subunits. The separation of light and heavy subunits of gamma-glutamyltransferase formed in reversed micelles was carried out and their catalytic properties were studied. The two subunits catalyze hydrolysis and transpeptidation reactions; autotranspeptidation reaction is detected only in the case of the heavy subunit. These findings imply that the reversed micelles of surfactants in organic solvents function as the matrices with adjustable size permitting to regulate the supramolecular structure and the catalytic activity of oligomeric enzymes.     

Fluorescence lifetimes in hydrated bovine serum albumin powders

The average relaxation time for tryptophan excited state decay increases progressively with water content in bovine serum albumin powders. A sharp lifetime increase is observed at low water coverage, followed by a slower increase at intermediate hydration levels. As the water content exceeds 0.5g H2O/g protein, the lifetime increase is again steep.     

Kinetic studies of fusarium solani pisi cutinase used in a gas/solid system: transesterification and hydrolysis reactions

Fusarium solani cutinase supported onto Chromosorb P was used to catalyze transesterification (alcoholysis) and hydrolysis on short volatile alcohols and esters in a continuous gas/solid bioreactor. In this system, a solid phase composed of a packed enzymatic preparation was continuously percolated with carrier gas which fed substrates and removed reaction products simultaneously. A kinetic study was performed under differential operating conditions in order to get initial reaction rates. The effect of the hydration state of the biocatalyst on the kinetics was studied for 3 conditions of hydration (a(w) = 0.2, a(w) = 0.4 and a(w) = 0.6), the alcoholysis of propionic acid methyl ester with n-propanol, and for 5 hydration levels (from a(w) = 0.2 to a(w) = 0.6) for the hydrolysis of propionic acid methyl, ethyl or propyl esters. F. solani cutinase was found to have an unusual kinetic behavior. A sigmoid relationship between the rate of transesterification and the activity of methyl propionate was observed, suggesting some form of cooperative activation of the enzyme by one of its substrate. For the hydrolysis of short volatile propionic acid alkyl esters, threshold effects on the reaction rate, highly depending on the water activity and the substrate polarity, are reported. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 1-8, 1997.     

Turnover of beta-galactosidase in fibroblasts from patients with genetically different types of beta-galactosidase deficiency.

The turnover of lysosomal beta-galactosidase was studied in fibroblast cultures from patients with Gm1-gangliosidosis and combined beta-galactosidase and neuraminidase deficiency, which had 5-10% residual beta-galactosidase activity. beta-Galactosidase was specifically inactivated with the suicide substrate beta-D-galactopyranosylmethyl-p-nitro-phenyltriazene (beta-Gal-MNT) and from the subsequent restoration of enzyme activity in cell cultures turnover times were calculated. By using [3H]beta-Gal-MNT, the hydrolytic activity per molecule of beta-galactosidase was determined. 3H-labelled beta-D-galactopyranosylmethylamine, the precursor of [3H]beta-gal-MNT, was obtained by Raney-nickel-catalysed exchange with 3H2O. The rate of synthesis of beta-galactosidase in normal and all mutant cells tested was found to be 0.4-0.5 pmol/day per mg of cellular protein. The GM1-gangliosidosis cells tested contain the normal amount of 0.5 pmol of beta-galactosidase/mg of protein with a normal turnover time of about 10 days, but only 10% of beta-galactosidase activity per enzyme molecule. Cells with combined beta-galactosidase and neuraminidase deficiency contain only 0.3 pmol of beta-galactosidase/mg of protein with a decreased turnover time of 1 day and normal hydrolytic properties (200 nmol of 4-methylumbelliferyl galactoside/h pmol of beta-galactosidase).     

α‐chymotrypsin in water–acetone and water–dimethyl sulfoxide mixtures: Effect of preferential solvation and hydration

We investigated water/organic solvent sorption and residual enzyme activity to simultaneously monitor preferential solvation/hydration of protein macromolecules in the entire range of water content at 25°C. We applied this approach to estimate protein destabilization/stabilization due to the preferential interactions of bovine pancreatic α‐chymotrypsin with water‐acetone (moderate‐strength H‐bond acceptor) and water‐DMSO (strong H‐bond acceptor) mixtures. There are three concentration regimes for the dried α‐chymotrypsin. α‐Chymotrypsin is preferentially hydrated at high water content. The residual enzyme activity values are close to 100%. At intermediate water content, the dehydrated α‐chymotrypsin has a higher affinity for acetone/DMSO than for water. Residual enzyme activity is minimal in this concentration range. The acetone/DMSO molecules are preferentially excluded from the protein surface at the lowest water content, resulting in preferential hydration. The residual catalytic activity in the water‐poor acetone is ～80%, compared with that observed after incubation in pure water. This effect is very small for the water‐poor DMSO. Two different schemes are operative for the hydrated enzyme. At high and intermediate water content, α‐chymotrypsin exhibits preferential hydration. However, at intermediate water content, in contrast to the dried enzyme, the initially hydrated α‐chymotrypsin possesses increased preferential hydration parameters. At low water content, no residual enzyme activity was observed. Preferential binding of DMSO/acetone to α‐chymotrypsin was detected. Our data clearly demonstrate that the hydrogen bond accepting ability of organic solvents and the protein hydration level constitute key factors in determining the stability of protein–water–organic solvent systems.     

Substrate specificity of an α-amino acid ester hydrolase produced by Acetobacter turbidans A.T.C.C. 9325

A partially purified preparation of an alpha-amino acid ester hydrolase was obtained from Acetobacter turbidans A.T.C.C. 9325, which catalyses synthesis of 7-(d-alpha-amino-alpha-phenylacetamido)-3-cephem-3-methyl-4- carboxylic acid (cephalexin) from methyl d-alpha-aminophenylacetate and 7-amino-3-deacetoxycephalosporanic acid. The enzyme preparation catalysed both cephalosprin synthesis from 7-amino-3-deacetoxycephalosporanic acid and suitable amino acid esters (e.g. methyl d-alpha-aminophenylacetate, l-cysteine methyl ester, glycine ethyl ester, d-alanine methyl ester, methyl dl-alpha-aminoiso-butyrate, l-serine methyl ester, d-leucine methyl ester, l-methionine methyl ester) and the hydrolysis of such esters. The substrate specificity of the enzyme preparation for the hydrolysis closely paralleled the acyl-donor specificity for cephalosporin synthesis, even to the reaction rates. Only alpha-amino acid derivatives could act as acyl donors. The hydrogen atom on the alpha-carbon atom was not always required by acyl donors. The hydrolysis rate was markedly diminished by adding 7-amino-3-deacetoxycephalosporanic acid to reaction mixtures, but no effect on the total reaction rate (the hydrolysis rate plus synthesis rate) was observed with various concentrations of 7-amino-3-deacetoxycephalosporanic acid. Both the hydrolytic and the synthetic activities of the enzyme preparation were inhibited by high concentrations of some acyl donors (e.g. methyl d-alpha-aminophenylacetate, ethyl d-alpha-aminophenylacetate). The enzyme preparation hydrolysed alpha-amino acid esters much more easily than alpha-amino acid derivatives with an acid-amide bond.     

Characterization of a novel lipolytic enzyme from Aspergillus oryzae

In this study, we report the characterization of a protein from Aspergillus oryzae, exhibiting sequence identity with paraben esterase from the genus Aspergillus. The coding region of 1,586 bp, including a 77-bp intron, encoded a protein of 502 amino acids. The gene without the signal peptide of 19 amino acids was cloned into a vector, pPICZαC, and expressed successfully in Pichia pastoris as an active extracellular protein. The purified recombinant protein had pH and temperature optima of 7.0–8.0 and 30 °C, respectively, and was stable at the pH range of 7.0–10.0 and up to 40 °C. The optimal substrate for hydrolysis by the purified recombinant protein, among a panel of α-naphthyl esters (C2–C16), was α-naphthyl butyrate (C4), with activity of 0.16 units/mg protein. The considerable hydrolytic activity of the purified recombinant enzyme toward tributyrin was determined. However, no paraben esterase activity was detected toward the ethyl, propyl, and butyl esters of 4-hydroxybenzoic acid. In addition, no activity was detected toward the methyl esters of ferulic, p-coumaric, caffeic, and sinapic acids that would indicate feruloyl esterase activity.     

Tsukamurella sp. E105 as a new biocatalyst for highly enantioselective hydrolysis of ethyl 2-(2-oxopyrrolidin-1-yl) butyrate

A new bacterial strain, E105, has been introduced as a biocatalyst for the enantioselective hydrolysis of ethyl (R,S)-2-(2-oxopyrrolidin-1-yl) butyrate, (R,S)-1, to (S)-2-(2-oxopyrrolidin-1-yl) butyric acid, (S)-2. This strain was isolated from 60 soil samples using (R,S)-1 as the sole carbon source. The isolate was identified as Tsukamurella tyrosinosolvens E105, based on its morphological characteristics, physiological tests, and 16S rDNA sequence analysis. The process of cell growth and hydrolase production for this strain was then investigated. The hydrolase activity reached its maximum after cultivation at 200?rpm and 30?°C for 36?h. Furthermore, the performance of the enantioselective hydrolysis of (R,S)-1 was studied. The optimal reaction temperature, initial pH, substrate concentration, and concentration of suspended cells were 30?°C, 6.8, 10 and 30?g/l (DCW), respectively. Under these conditions, a high conversion (>45?%) of the product (S)-2 with an excellent enantiomeric excess (ee) (>99?%), and a satisfied enantiomeric ratio (E) (>600) as well were obtained. This study showed that the bacterial isolate T. tyrosinosolvens E105 displayed a high enantioselectivity towards the hydrolysis of racemic ethyl 2-(2-oxopyrrolidin-1-yl) butyrate.     

Hydrolysis of beta-galactosyl ester linkage by beta-galactosidases

p-Hydroxybenzoyl beta-galactose (pHB-Gal) was synthesized chemically to examine the hydrolytic activity of beta-galactosyl ester linkage by beta-galactosidases. The enzyme from Penicillium multicolor hydrolyzed the substrate as fast as p-nitrophenyl beta-galactoside (pNP-Gal), a usual substrate with a beta-galactosidic linkage. The enzymes from Escherichia coli and Aspergillus oryzae hydrolyzed pHB-Gal with almost the same rates as pNP-Gal. The enzymes from Bacillus circulans, Saccharomyces fragilis, and bovine liver showed much lower activities. pH-activity profiles, inhibition analysis, and kinetic properties of the enzymic reaction on pHB-Gal suggested that beta-galactosidase had only one active site for hydrolysis of both galactosyl ester and galactoside. The Penicillium enzyme hydrolyzed pHB-Gal in the presence of H218O to liberate galactose containing 18O. This result suggests the degradation occurs between the anomeric carbon and an adjacent O atom in the ester linkage of pHB-Gal.     

The kinetic mechanism of yeast inorganic pyrophosphatase

The kinetic mechanism of yeast inorganic pyrophosphatase (PPase) was examined by carrying out initial velocity studies. Ca2+ and Rh(H2O)4(methylenediphosphonate) (Rh(H2O)4PCP) were used as dead-end inhibitors to study the order of binding of Cr(H2O)4PP to the substrate site and Mg2+ to the "low affinity" activator site on the enzyme. Competitive inhibition was observed for Ca2+ vs Mg2+ (Kis = 0.93 +/- 0.03 mM), for Rh(H2O)4PCP vs Cr(H2O)4PP (Kis = 0.25 +/- 0.07 mM), and for RH(H2O)4PCP vs Mg2+ (Kis = 0.38 +/- 0.03 mM). Uncompetitive inhibition was observed for Ca2+ vs Cr(H2O)4PP (Kii = 0.49 +/- 0.01). On the basis of these results a rapid equilibrium ordered mechanism in which Cr(H2O)4PP binding precedes Mg2+ ion binding is proposed. The inert substrate analog, Mg(imidodiphosphate) (MgPNP) was shown to induce Mg2+ inhibition of the PPase-catalyzed hydrolysis of MgPP. The Mg2+ inhibition observed was competitive vs MgPP and partial. These results suggest that Mg2+/MgPNP release from the enzyme occurs in preferred rather than strict order and that the Mg2+/MgPP-binding steps are at steady state. Zn2+, Co2+, and Mn2+ (but not Mg2+) displayed activator inhibition of the PPase-catalyzed hydrolysis of PPi (this study) and of Cr(H2O)4PP (W.B. Knight, S. Fitts, and D. Dunaway-Mariano, (1981) Biochemistry 20, 4079). These findings suggest that cofactor release from the low affinity cofactor site on the enzyme must precede product release and that Zn2+, Mn2+, and Co2+ (but not Mg2+) have high affinities for the cofactor sites on both the PPase.M.MPP and PPase.M.M(P)2 complexes. The role of the metal cofactor in determining PPase substrate specificity was briefly explored by testing the ability of the Mg2+ complex of tripolyphosphate (PPPi) (a substrate for the Zn2+-activated enzyme but not the Mg2+-activated enzyme) to induce Mg2+ inhibition of PPase-catalyzed hydrolysis of MgPP. MgPPP was shown to be as effective as MgPNP in inducing competitive Mg2+ inhibition (vs MgPP). This result suggests that the low affinity Mg2+ cofactor-binding site present in the enzyme-MgPP complex is maintained in the enzyme-MgPPP complex. Thus, failure of Mg2+ to bind to the enzyme-MgPPP complex was ruled out as a possible explanation for the failure of the Mg2+-activated enzyme to catalyze the hydrolysis of MgPPP.     

Plant lipases: biocatalyst aqueous environment in relation to optimal catalytic activity in lipase-catalyzed synthesis reactions.

Adsorption and desorption isotherms of two commercial enzyme preparations of papain and bromelain were determined with a Dynamic Vapor System. The Guggenheim-Anderson-deBoer (GAB) modeling of the obtained sorption isotherms allowed the definition of different levels of hydration of those samples. Afterward, these enzyme preparations were used as biocatalysts in water and solvent-free esterification and alcoholysis reactions. The evolution of the obtained fatty acid ester level as a function of the initial hydration level of the biocatalyst, i.e., thermodynamic water activity (a(w)) and water content, was studied. The results show an important correlation between the initial hydration level of the biocatalyst and its catalytic activity during the lipase-catalyzed synthesis reactions. Thus, the Carica papaya lipase (crude papain preparation) catalytic activity is highly dependent on the biocatalyst hydration state. The optimized synthesis reaction yield is obtained when the a(w) value of the enzyme preparation is stabilized at 0.22, which corresponds to 2% water content. This optimal level of hydration occurs on the linear part of the biocatalyst's sorption isotherm, where the water molecules can form a mono- or multiple layer with the protein network. The synthesis reaction yield decreases when the a(w) of the preparation is higher than 0.22, because the excess water molecules modify the system equilibrium leading to the reverse and competitive reaction, i.e., hydrolysis. These results show also that an optimal storage condition for the highly hydrophilic crude papain preparation is a relative humidity strictly lower than 70% to avoid an irreversible structural transition leading to a useless biocatalyst. Concerning the bromelain preparation, no effect of the hydration level on the catalytic activity during esterification reactions was observed. This biocatalyst has too weak a catalytic activity which makes it difficult to observe any differences. Furthermore, the bromelain preparation is far more hydrophobic as it adsorbs only 18 g of water per 100 g of dry material at a(w) around 0.90. No deliquescence of this enzymatic preparation is observed at this a(w) value.     

Solvent isotope effects for lipoprotein lipase catalyzed hydrolysis of water-soluble p-nitrophenyl esters

Solvent deuterium isotope effects on the rates of lipoprotein lipase (LpL) catalyzed hydrolysis of the water-soluble esters p-nitrophenyl acetate (PNPA) and p-nitrophenyl butyrate (PNPB) have been measured and fall in the range 1.5-2.2. The isotope effects are independent of substrate concentration, LpL stability, and reaction temperature and hence are effects on chemical catalysis and not due to a medium effect of D2O on LpL stability and/or conformation. pL (L = H or D) vs. rate profiles for the Vmax/Km of LpL-catalyzed hydrolysis of PNPB increase sigmoidally with increasing pL. Least-squares analysis of the profiles gives pKaH2O = 7.10 +/- 0.01, pKaD2O = 7.795 +/- 0.007, and a solvent isotope effect on limiting velocity at high pL of 1.97 +/- 0.03. Because the pL-rate profiles are for the Vmax/Km of hydrolysis of a water-soluble substrate, the measured pKa's are intrinsic acid-base ionization constants for a catalytically involved LpL active-site amino acid side chain. Benzeneboronic acid, a potent inhibitor of LpL-catalyzed hydrolysis of triacylglycerols [Vainio, P., Virtanen, J. A., & Kinnunen, P. K. J. (1982) Biochim. Biophys. Acta 711, 386-390], inhibits LpL-catalyzed hydrolysis of PNPB, with Ki = 6.9 microM at pH 7.36, 25 degrees C. This result and the solvent isotope effects for LpL-catalyzed hydrolysis of water-soluble esters are interpreted in terms of a proton transfer mechanism that is similar in many respects to that of the serine proteases.     

An NMR method to characterize multiple water compartments on mammalian collagen

A molecular model is proposed to explain water 1H NMR spin-lattice relaxation at different levels of hydration (NMR titration method) on collagen. A fast proton exchange model is used to identify and characterize protein hydration compartments at three distinct Gibbs free energy levels. The NMR titration method reveals a spectrum of water motions with three well-separated peaks in addition to bulk water that can be uniquely characterized by sequential dehydration. Categorical changes in water motion occur at critical hydration levels h (g water/g collagen) defined by integral multiples N = 1, 4 and 24 times the fundamental hydration value of one water bridge per every three amino acid residues as originally proposed by Ramachandran in 1968. Changes occur at (1) the Ramachandran single water bridge between a positive amide and negative carbonyl group at h1 = 0.0658 g/g, (2) the Berendsen single water chain per cleft at h2 = 0.264 g/g, and (3) full monolayer coverage with six water chains per cleft level at h3 = 1.584 g/g. The NMR titration method is verified by comparison of measured NMR relaxation compartments with molecular hydration compartments predicted from models of collagen structure. NMR titration studies of globular proteins using the hydration model may provide unique insight into the critical contributions of hydration to protein folding.     

Stereochemistry of D-galactal and D-galacto-octenitol hydration by coffee bean alpha-galactosidase: insight into catalytic functioning of the enzyme.

Green coffee bean alpha-galactosidase was found to catalyze the hydration of D-galactal and (Z)-3,7-anhydro-1,2-dideoxy-D-galacto-oct-2-enitol (D-galacto-octenitol), each a known substrate for beta-galactosidase. The hydration of D-galactal by the alpha-galactosidase in D2O yielded 2-deoxy-2(S)-D-[2-2H]galactose; the hydration of D-[2-2H]galacto-octenitol in H2O yielded 1,2-dideoxy-2(R)-D-[2-2H]galactooct-3-ulose. Thus, the enzyme protonated each substrate from beneath the plane of the ring, as assumed for alpha-D-galactosides. These results provide an unequivocal assignment of the orientation of an acidic catalytic group to the alpha-galactosidase reaction center. In addition, they reveal a pattern of glycal/exocyclic enitol/glycoside protonation by the enzyme that differs from the pattern reported for beta-galactosidase and from that reported for alpha-glucosidases. Further findings show that D-galacto-octenitol is hydrated by the coffee bean alpha-galactosidase to form the alpha-anomer of 1,2-dideoxy-D-galactooctulose and by Escherichia coli beta-galactosidase to form the beta-anomer. That each enzyme converts this enolic substrate to a product whose de novo anomeric configuration matches that formed from its D-galactosidic substrates provides new evidence for the role of protein structure in controlling the steric outcome of reactions catalyzed by these and other glycosylases. The findings are discussed in light of the concept that catalysis by glycosidases involves a "plastic" protonation phase and a "conserved" product configuration phase.     

L-citrulline production from L-arginine by macrophage nitric oxide synthase. The ureido oxygen derives from dioxygen.

Previously proposed mechanisms for the production of L-citrulline from L-arginine by macrophage nitric oxide (NO.) synthase involve either hydrolysis of arginine or hydration of an intermediate and thus predict incorporation of water oxygen into L-citrulline. Macrophage NO. synthase was incubated with L-arginine, NADPH, tetrahydrobiopterin, FAD, and dithiothreitol in H2(18)/16O2. L-Citrulline produced in this reaction was analyzed with gas chromatography/mass spectrometry. Its mass spectrum matched that of L-citrulline generated in H2(16)O/16O2. The base fragment ion of m/z 99 was shown to contain the ureido carbonyl group by using L-[guanidino-13C]arginine as substrate. When the enzyme reaction was performed in H2(16)O/18O2, the base fragment ion shifted to m/z 101 with L-[guanidino-12C]arginine as the substrate and to m/z 102 with L-[guanidino-13C]arginine. These results indicate that the ureido oxygen of the L-citrulline product of macrophage NO.synthase derives from dioxygen and not from water.     

Water of hydration in the intra- and extra-cellular environment of human erythrocytes

The proton nuclear magnetic resonance (NMR) titration method (which requires measurement of the relaxation rate at multiple measured levels of dehydration) was applied to the analysis of human erythrocytes, a hemoglobin solution, plasma, and serum. The results allowed identification of bulk water and four motionally perturbed water of hydration subfractions. Based on previous NMR studies of homopolypeptides we designated these subfractions as superbound, irrotationally bound, rotationally bound, and structured. The total water of hydration (sum of both structured and bound water subfractions) in plasma, serum, and hemoglobin ranged from 2.78 to 3.77 g H2O/g dry mass and the sum of the three bound water subfractions ranged from 1.23 to 1.72 g H2O/g dry mass. The total water of hydration on hemoglobin, as determined by (i) spin-lattice (T1) and spin-spin (T2) NMR data, (ii) quench ice-crystal imprint size, (iii) calculations based on osmotic pressure data, and (iv) two other methods, ranged from 2.26 to 3.45 g H2O/g dry mass. In contrast, the estimates of total water of hydration in the intact erythrocytes ranged from 0.34 to 1.44 g H2O/g dry mass, as determined by osmotic activity and spin-lattice titration, respectively. Studies on the magnetic-field dependence of the spin-lattice relaxation rate (1/T1 rho) of solvent water nuclei in protein solutions and in intact and disrupted erythrocytes indicated that hemoglobin aggregation exists in the intact erythrocytes and that erythrocyte disruption decreases the extent of hemoglobin aggregation. Together, the present and past data indicate that the extent of water of hydration associated with hemoglobin depends on the amount of salt present and the degree of aggregation of the hemoglobin molecules.