Protein stabilization via hydrophilization. Covalent modification of trypsin and alpha-chymotrypsin

This paper experimentally verifies the idea presented earlier that the contact of nonpolar clusters located on the surface of protein molecules with water destabilizes proteins. It is demonstrated that protein stabilization can be achieved by artificial hydrophilization of the surface area of protein globules by chemical modification. Two experimental systems are studied for the verification of the hydrophilization approach. The surface tyrosine residues of trypsin are transformed to aminotyrosines using a two-step modification procedure: nitration by tetranitromethane followed by reduction with sodium dithionite. The modified enzyme is much more stable against irreversible thermoinactivation: the stabilizing effect increases with the number of aminotyrosine residues in trypsin and the modified enzyme can become even 100 times more stable than the native one. Alpha-chymotrypsin is covalently modified by treatment with anhydrides or chloroanhydrides of aromatic carboxylic acids. As a result, different numbers of additional carboxylic groups (up to five depending on the structure of the modifying reagent) are introduced into each Lys residue modified. Acylation of all available amino groups of alpha-chymotrypsin by cyclic anhydrides of pyromellitic and mellitic acids results in a substantial hydrophilization of the protein as estimated by partitioning in an aqueous Ficoll-400/Dextran-70 biphasic system. These modified enzyme preparations are extremely stable against irreversible thermal inactivation at elevated temperatures (65-98 degrees C); their thermostability is practically equal to the stability of proteolytic enzymes from extremely thermophilic bacteria, the most stable proteinases known to date.     

Chemical Modification of Specific Active Site Amino Acid Residues of Enterobacter aerogenes Glycerol Dehydrogenase

Enterobacter aerogenes glycerol dehydrogenase (GlDH EC 1.1.1.6), a tetrameric NAD + specific enzyme catalysing the interconversion of glycerol and dihydroxyacetone, was inactivated on reaction with pyridoxal 5′-phosphate (PLP) and o -phthalaldehyde (OPA). Fluorescence spectra of PLP-modified, sodium borohydride-reduced GlDH indicated the specific modification of ? -amino groups of lysine residues. The extent of inhibition was concentration and time dependent. NAD + and NADH provided complete protection against enzyme inactivation by PLP, indicating the reactive lysine is at or near the coenzyme binding site. Modification of GlDH by the bifunctional reagent OPA, which reacts specifically with proximal ? -NH 2 group of lysines and -SH group of cysteines to form thioisoindole derivatives, inactivated the enzyme. Molecular weight determinations of the modified enzyme indicated the formation of intramolecular thioisoindole formation. Glycerol partially protected the enzyme against OPA inactivation, whereas NAD + was ineffective. These results show that the lysine involved in the OPA reaction is different from the PLP-reactive lysine, which is at or near the coenzyme binding site. DTNB titration showed the presence of only a single cysteine residue per monomer of GlDH. This could be participating with a proximal lysine residue to form a thioisoindole derivative observed as a result of OPA modification.     

2-Bromoacetylaminopentitol 1,5-bisphosphate as an affinity label for ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

2-Bromoacetylaminopentitol 1,5-bisphosphate (BrAcNH-pentitol-P2) (an epimeric mixture of 2-bromoacetylamino-2-deoxy-D-ribitol bisphosphate and 2-bromoacetylamino-2-deoxy-D-arabinitol 1,5-bisphosphate) has been synthesized from D-ribulose 1,5-bisphosphate by reductive amination with sodium cyanoborohydride followed by bromoacetylation of the resultant amine with bromoacetyl bromide. Under conditions that favor full activation of the enzyme, ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum is completely inactivated by BrAcNH-pentitol-P2 in a pseudo-first order process. A rate saturation is observed with a minimal inactivation half-life of 38 min and Kinact for reagent of 0.38 mM. The competitive inhibitor 2-carboxyribitol 1,5-bisphosphate reduces the rate of inactivation, and kinetic analyses are consistent with the protection reflecting true competition of inhibitor and reagent for the same site. As shown with isotopically labeled reagent, complete inactivation is associated with covalent incorporation of 1.1 mol of reagent/mol of subunit. Based on reversibility of inactivation by thiolysis and based on analysis of labeled products in acid hydrolysates of the modified enzyme, a methionyl sulfonium salt is the reaction product. In the absence of CO2 and Mg2+ (ligands required for activation), the enzyme is resistant to BrAcNH-pentitol-P2, which suggests that the site-specific modification of a methionyl residue requires a fully functional catalytic center.     

Chemical modification of specific active site amino acid residues of Enterobacter aerogenes glycerol dehydrogenase

Enterobacter aerogenes glycerol dehydrogenase (G1DH EC 1.1.1.6), a tetrameric NAD+ specific enzyme catalysing the interconversion of glycerol and dihydroxyacetone, was inactivated on reaction with pyridoxal 5-phosphate (PLP) and o-phthalaldehyde (OPA). Fluorescence spectra of PLP-modified, sodium borohydride-reduced G1DH indicated the specific modification of epsilon-amino groups of lysine residues. The extent of inhibition was concentration and time dependent. NAD+ and NADH provided complete protection against enzyme inactivation by PLP, indicating the reactive lysine is at or near the coenzyme binding site. Modification of G1DH by the bifunctional reagent OPA, which reacts specifically with proximal epsilon-NH2 group of lysines and -SH group of cysteines to form thioisoindole derivatives, inactivated the enzyme. Molecular weight determinations of the modified enzyme indicated the formation of intramolecular thioisoindole formation. Glycerol partially protected the enzyme against OPA inactivation, whereas NAD+ was ineffective. These results show that the lysine involved in the OPA reaction is different from the PLP-reactive lysine, which is at or near the coenzyme binding site. DTNB titration showed the presence of only a single cysteine residue per monomer of G1DH. This could be participating with a proximal lysine residue to form a thioisoindole derivative observed as a result of OPA modification.     

Inactivation of pyrophosphate-dependent phosphofructokinase from Propionibacterium freudenreichii by pyridoxal 5'-phosphate. Determination of the pH dependence of enzyme-reactant dissociation constants from protection against inactivation

The pyrophosphate-dependent phosphofructokinase from Propionibacterium freudenreichii is rapidly inactivated by low concentrations of pyridoxal 5'-phosphate (PLP). The inactivation is first order with respect to PLP and the rate increases linearly with PLP concentrations suggesting that over the concentration range used no significant E-PLP complex accumulates during inactivation. The rate of inactivation decreases at high and low pH and this is discussed in terms of the mechanism of Schiff base formation. The presence of any reactants decreases the rate of inactivation to 0 at infinite concentration. This protection against inactivation has been used to obtain the pH dependence of the dissociation constants of all enzyme-reactant binary complexes. Reduction of the PLP-inactivated enzyme with NaB[3H]4 indicates that about 7 lysines are modified in free enzyme and fructose 6-phosphate protects 2 of these from modification. The pH dependence of the enzyme-reactant dissociation constants suggests that the phosphates of fructose 6-phosphate, fructose 1,6-bisphosphate, inorganic phosphate, and Mg-pyrophosphate must be completely ionized and that lysines are present in the vicinity of the 1- and 6-phosphates of the sugar phosphate and bisphosphate probably directly coordinated to these phosphates.     

Inactivation of yeast hexokinase by o-phthalaldehyde: evidence for the presence of a cysteine and a lysine at or near the active site

Yeast hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1), a homodimer, was rapidly and irreversibly inactivated by o-phthalaldehyde at 25 degrees C (pH 7.3). The reaction followed pseudo-first-order kinetics over a wide range of the inhibitor concentration. The second-order-rate constant for the inactivation of hexokinase was estimated to be 45 M-1.s-1. Hexokinase was protected more by sugar substrates than by nucleoside triphosphates during inactivation by o-phthalaldehyde. Absorption spectrum (lambda max 338 nm), and fluorescence excitation (lambda max 363 nm) and emission (lambda max 403 nm) spectra of the hexokinase-o-phthalaldehyde adduct were consistent with the formation of an isoindole derivative. These results also suggest that sulfhydryl and epsilon-amino functions of the cysteine and lysine residues, respectively, participating in the isoindole formation are about 3 A apart in the native enzyme. About 2 mol of the isoindole per mol of hexokinase dimer were formed following complete loss of the phosphotransferase activity. Chemical modification of hexokinase by iodoacetamide in the presence of mannose resulted in the modification of six sulfhydryl groups per mol of hexokinase with retention of the phosphotransferase activity. Subsequent reaction of the iodoacetamide modified hexokinase with o-phthalaldehyde resulted in complete loss of the phosphotransferase activity with concomitant modification of the remaining two sulfhydryl groups of hexokinase. Chemical modification of hexokinase by iodoacetamide in the absence of mannose resulted in complete inactivation of the enzyme. The iodoacetamide inactivated hexokinase failed to react with o-phthalaldehyde as evidenced by the absence of a fluorescence emission maximum characteristic of the isoindole derivative. The holoenzyme failed to react with [5'-(p-fluorosulfonyl)benzoyl]adenosine. The dissociated hexokinase could be inactivated by [5'-(p-fluorosulfonyl)benzoyl]adenosine; the degree of inactivation paralleled the extent of reaction between o-phthalaldehyde and the nucleotide-analog modified enzyme. Thus, it is concluded that two cysteines and lysines at or near the active site of the hexokinase were involved in reaction with o-phthalaldehyde following complete loss of the phosphotransferase activity. An important finding of this investigation is that the lysines, involved in isoindole formation, located at or near the active site are probably buried.(ABSTRACT TRUNCATED AT 400 WORDS)     

Physicochemical evidence for the existence of two pyridoxal 5'-phosphate binding sites on glutamate dehydrogenase and characterization of their functional role.

Kinetic studies of pyridoxal 5'-phosphate binding to glutamate dehydrogenase (EC 1.4.1.3) has provided evidence for two specific binding sites, chemically identified as Lys 126 and Lys 333. Use of protecting ligands permitted the selective modification of only one of these lysines, and showed that (1) Lys 333 modification results in depolymerisation of the enzyme into active hexamers; (2) Lys 126-modified enzyme was 92% inactivated. The residual activity was desensitized to GTP. The inactivation process was cooperative, maximum inactivation occurring as soon as half of the Lys 126 were modified.     

Tyramine-modified pectins via periodate oxidation for soybean hull peroxidase induced hydrogel formation and immobilization

Pectin was modified by oxidation with sodium periodate at molar ratios of 2.5, 5, 10, 15 and 20 mol% and reductive amination with tyramine and sodium cyanoborohydride afterwards. Concentration of tyramine groups within modified pectin ranged from 54.5 to 538 μmol/g of dry pectin while concentration of ionizable groups ranged from 3.0 to 4.0 mmol/g of dry polymer compared to 1.5 mmol/g before modification due to the introduction of amino group. All tyramine-pectins showed exceptional gelling properties and could form hydrogel both by cross-linking of carboxyl groups with calcium or by cross-linking phenol groups with peroxidase in the presence of hydrogen peroxide. These hydrogels were tested as carriers for soybean hull peroxidase (SHP) immobilization within microbeads formed in an emulsion based enzymatic polymerization reaction. SHP immobilized within tyramine-pectin microbeads had an increased thermal and organic solvent stability compared to the soluble enzyme. Immobilized SHP was more active in acidic pH region and had slightly decreased K _m value of 2.61 mM compared to the soluble enzyme. After 7 cycles of repeated use in batch reactor for pyrogallol oxidation microbeads, immobilized SHP retained half of the initial activity.

     

Mechanism of action of cutinase: chemical modification of the catalytic triad characteristic for serine hydrolases

Cutinase from Fusarium solani f. sp. pisi was inhibited by diisopropyl fluorophosphate and phenylboronic acid, indicating the involvement of an active serine residue in enzyme catalysis. Quantitation of the number of phosphorylated serines showed that modification of one residue resulted in complete loss of enzyme activity. One essential histidine residue was modified with diethyl pyrocarbonate. This residue was buried in native cutinase and became accessible to chemical modification only after unfolding of the enzyme by sodium dodecyl sulfate. The modification of carboxyl groups with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide in the absence of sodium dodecyl sulfate did not result in inactivation of the enzyme; however, such modifications in the presence of sodium dodecyl sulfate resulted in complete loss of enzyme activity. The number of residues modified was determined by incorporation of [14C]glycine ethyl ester. Modification of cutinase in the absence of sodium dodecyl sulfate and subsequent unfolding of the enzyme with detergent in the presence of radioactive glycine ester showed that one buried carboxyl group per molecule of cutinase resulted in complete inactivation of the enzyme. Three additional peripheral carboxyl groups were modified in the presence of sodium dodecyl sulfate. Carbethoxylation of the essential histidine and subsequent incubation with the esterase substrate p-nitrophenyl [1-14C]acetate revealed that carbethoxycutinase was about 10(5) times less active than the untreated enzyme. The acyl-enzyme intermediate was stabilized under these conditions and was isolated by gel permeation chromatography. The results of the present chemical modification study indicate that catalysis by cutinase involves the catalytic triad and an acyl-enzyme intermediate, both characteristic for serine proteases.     

Functional and structural responses of liver alcohol dehydrogenase to lysine modifications

Acetylation, glycosylation, and methylation, which modify lysine residues of horse liver alcohol dehydrogenase, have been investigated. Acetylation reacted with approximately two-third of the total lysines to induce the greatest structural changes of the enzyme. Glycosylation modified only one lysine residue selectively with indiscernible structural changes. The glycosylation effect was very specific with respect to diastereoisomers for aldopentoses, aldohexoses, and ketohexoses. Methylation produced the largest enhancement in the oxidative activity, which is related to the stability of the modified enzyme to prolonged modification and thermal denaturation. Kinetic studies revealed that a change in the maximal velocity was primarily responsible for the observed activity differences in the modifications.     

静电作用与修饰铜锌超氧化物歧化酶的稳定性

铜锌超氧化物歧化酶(Cu, Zn-SOD)表面的赖氨酸经化学修饰后, 酶的稳定性显著提高. 赖氨酸被修饰后, 酶的电荷结构遂发生变化, 从而影响到酶分子电场. 使用FDPB方法(有限差分法求解Poission-Boltzman方程)计算了酶修饰前后的静电场变化, 以及对维持酶的结构稳定起重要作用的Cu, Zn配位结构的影响.结果表明, Cu, Zn配位体的两级离解常数在酶修饰后分别约下降10³, 10⁶.     

Modification and inactivation of rhodanese by 2,4,6-trinitrobenzenesulphonic acid

Bovine liver rhodanese (thiosulphate sulphurtransferase, EC 2.8.1.1) is modified by 2,4,6-trinitrobenzenesulphonic acid, by the use of modifying agent concentrations in large excess over enzyme protein concentration. The end-point of the reaction, viz., the number, n, per enzyme protein molecule, of modifiable amino groups was determined graphically by the Kézdy-Swinbourne procedure. It was found that the value for n depends on the pH of the reaction medium, and ranges from 2, at pH 7.00, to 10.66, at pH 9.00. Again, the value for n increases with an increase in the concentration of 2,4,6-trinitrobenzenesulphonic acid used, with values ranging from 3.52, at 0.10 mM modifying agent, to 8.96, at 2 mM modifying agent. Rhodanese primary amino groups modification by 2,4,6-trinitrobenzenesulphonic acid is described by a summation of exponential functions of reaction time at pH values of 8.00 or higher, while at lower pH values it is described by a single exponential function of reaction time. However, the log of the first derivative, at initial reaction conditions, of the equation describing protein modification, is found to be linearly dependent on the pH of the reaction. An identical linear dependence is also found when the log of the first derivative, at the start of the reaction, of the equation describing modification-induced enzyme inactivation is plotted against the pH values of the medium used. In consequence, the fractional concentration of rhodanese modifiable amino groups essential for enzyme catalytic function is equal to unity at all reaction pH values tested. It is accordingly concluded that, when concentrations of 2,4,6-trinitrobenzenesulphonic acid in excess of protein concentration are used, all rhodanese modifiable amino groups are essential for enzyme activity. A number of approaches were used in order to establish a mechanism for the modification-induced enzyme inactivation observed. These approaches, all of which proved to be negative, include the possible modification of enzyme sulfhydryl groups, disulphide bond formation, enzyme inactivation due to sulphite released during modification, modification-induced enzyme protein polymerization, syncatalytic enzyme modification and hydrogen peroxide-mediated enzyme inactivation.     

Chemical modification in situ of Escherichia coli 30 S ribosomal proteins by the site-specific reagent pyridoxal phosphate. Inactivation of the aminoacyl-tRNA and mRNA binding sites

epsilon-Amino groups of lysines of 30 S ribosomal subunits with affinity for phosphate groups were selectively modified in situ by reaction with pyridoxal phosphate and reduction of the Schiff base with nonradioactive or radioactive sodium borohydride. This reaction modified only a limited number of ribosomal proteins and resulted in the loss of only some 30 S activities. The modified proteins were identified and the extent of their modification determined. The main targets of the reaction were S3 greater than S1 greater than S6. The activity most severely affected by the pyridoxal phosphate reaction was mRNA-dependent aminoacyl-tRNA binding. Some inhibition of poly(U) binding was also observed, while neither binding of initiation factors nor association with 50 S subunits was inhibited. The inhibition of aminoacyl-tRNA binding showed distinct selectivity: the inhibition was far greater with NAcPhe-tRNA than with fMet-tRNA and with "A" site than with "P" site binding. In addition, initiation complex formation with some mRNAs (e.g. MS2 RNA) was affected more than with others (e.g. T7 early mRNA). Ribosome reconstitution experiments showed that the modification of protein S3 was the primary cause of the inhibition; a role was also played by ribosomal proteins S1, S2, and S21. Substrate protection experiments showed that the 30 S activity can be protected from pyridoxal phosphate inactivation upon formation of a ternary complex with poly(U) and tRNAPhe or NAcPhe-tRNAPhe. Accordingly, the extent of modification of ribosomal protein S3 was reduced in the ternary complex while modification of S1 was reduced in the presence of poly(U) alone.     

Improved thermal stability and activity in the cold-adapted lipase B from Candida antarctica following chemical modification with oxidized polysaccharides

In order to improve the thermal stability (t_1/2) and activity of lipase B from cold-adapted Candida antarctica (CALB), amino groups of the enzyme were chemically linked to a range of oxidized polysaccharides using a range of reducing agents. By chemically modifying CALB using 0.1% dextran (250 kDa) at pH 8.6 for 10 days using borane–pyridine complex as reducing agent, increased thermal stability (t_1/2, 168 min at 70°C) and activity (65% higher specific activity) was achieved compared to the unmodified enzyme (t_1/2, 18 min at 70°C). Improvements in thermostability were generally better with high molecular weight polymers such as dextran (40 and 250 kDa) or ficoll (70 and 400 kDa) in comparison to low molecular weight inulin (5 kDa). The shape of the polymer also appeared to be important with elongated, elipsoidal-shaped dextran providing better thermostabilization than spherical-shaped ficoll. Borane–pyridine complex was found to be a good, non-toxic reducing agent for improving thermostability, compared with sodium borohydride and sodium cyanoborohydride. An interesting finding was that, in all cases, specific activity of the modified enzymes increased with a concomitant increase in thermostability. This response defies the general principle of a trade-off between activity and stability, and demonstrates that chemical modification provides new avenues for improving the thermal stability of enzymes from psychrophiles without sacrificing their activity.     

Studies on phosphoglyceromutase from chicken breast muscle: chemical modification of lysyl residues.

To examine the role of lysyl residues in the activity of the enzyme, phosphoglyceromutase (PGM) from chicken breast muscle was chemically modified with trinitrobenzenesulfonate (TNBS) and pyridoxal 5'-phosphate. Trinitrophenylation resulted in modification of about nine lysines per mole of PGM with almost complete activity loss. Substrate (3-PGA) offered some protection to TNBS inactivation but cofactor (2,3-DPGA) did not. Reduction of the Schiff's base complex between pyridoxal 5'-phosphate and PGM gave irreversible inactivation of the enzyme. Inactivation was due to incorporation of 1 mol of pyridoxal 5'-phosphate per mole of PGM dimer through the epsilon-amino group of a lysyl residue. The effect of pyridoxal 5'-phosphate was specific for intact native enzyme and reaction with only one lysine per dimer was not due to induced conformational changes nor to dissociation of the reacted enzyme. 3-PGA prevented much of the reaction with pyridoxal 5'-phosphate with preservation of 70% of the activity and was a competitive inhibitor of the active site directed reagent. Cofactor (2,3-DPGA) acting noncompetitively, reduced the rate at which inactivation occurred with pyridoxal 5'-phosphate. Incorporation of 2,3-[32P]DPGA into PGM irreversibly inactivated with pyridoxal 5'-phosphate and NaBH4 was incomplete indicating hindrance to phosphorylation in the modified enzyme. The results indicate that a lysyl residue is located at or near the active site of PGM and that it is probably involved in the binding of 3-PGA.     

Thermal stabilization of amylolytic enzymes by covalent coupling to soluble polysaccharides

The immobilization of pullulanase and beta-amylase on soluble polysaccharides (dextrans and amylose) has been carried out. The method used for coupling the enzymes to the carbohydrate support involves limited periodate oxidation of the polysaccharide followed by reductive alkylation with sodium cyanoborohydride or borohydride. The influence of the degree of functionalization of the carbohydrate, the incubation time, the nature of the reducing agent and, for the dextrans studied, their molecular weight, on the properties of the conjugate were studied. We have observed an apparent correlation between the molecular weight of the glycoprotein conjugates formed and their thermal stability, resistance to urea denaturation and their kinetic parameters. By selecting the proper experimental conditions leading to conjugates with maximum thermal stabilities, it has also been shown that beta-amylase conjugates can hydrolyze starch at a temperature 20 degrees C higher than the corresponding value for the native enzyme. This result demonstrates that conjugation may result in modified enzymes leaving a high operational stability at elevated temperatures. We suggest that the immobilization method presented in this article represents an approach to the stabilization of enzymes employed at an industrial level, which may be of general application.     

New Methods for Chemo-Enzymatic Galactosidation of 2S Rapeseed Protein

Two chemo-enzymatic methodologies to synthesize neoglycoproteins from rapeseed 2S protein (napin) were developed. In the first approach, glycosidases were used to catalyse 1-O-glycosylation of serine residues, whereas in the second one, 6-N-galactosylation was examined using an amino-reduction reaction between the epsilon-NH2 of lysine residues and 6-oxogalactosides (readily available by means of the oxidation reaction of the corresponding galactosides mediated by galactose oxidase). Our results indicated that glycosidases were unable to glycosylate native proteins. Conversely, this reaction was possible, although in low yields (10%), after the introduction of a hydroxyethylene spacer. The latter modified proteins were obtained via the condensation of epsilon-NH2 of lysines with ethylene carbonate in basic medium (40% yield). The second approach was much more efficient, as 61% of the lysine residues were shown to be 6-N-galactosylated using sodium cyanoborohydride as a reduction reagent.     

Inactivation of Lysozyme By Alkylperoxyl Radicals

Thermolysis of 2, 2'-azo-bis-(2-amidinopropane) under air in the presence of lysozyme leads to extensive inactivation of the enzyme. The number of inactivated enzyme molecules per radical produced increases with the enzyme concentration up to values considerably larger than one. Enzyme inacitivation is accompanied by extensive tryptophan modification. Over the enzyme concentration range considered (1.7 to 130μM) nearly 4 tryptophan groups are modified per enzyme molecule inactivated. Both the inactivation and tryptophan modification are prevented by micromolar concentrations of propyl gallate. The results are interpreted in terms of an efficient inactivation of the enzyme by the alkylperoxyl radicals generated by thermolysis of the azocompound.     

Effect of the degree of modification of trypsin amino groups with polymers on the enzymatic and conformation properties of the protein

Trypsin was modified by introducing fragments containing an azo-bond into its molecule by the reaction of free amino groups of the enzyme with an azide of 2,2'-azobisisobutryic acid. Subsequently free-radical polymerization of N-vinyl pyrrolidone was carried out with the high molecular weight initiator obtained. The degree of modification of amino groups in trypsin was n = 6 divided by 12, which distinguishes this type of modification from that earlier proposed by the authors. In that case dichlorohydrate of dimethylimidate of 2,2'-azobisisobutyric acid was used for introducing azo-bonds into the molecule of the protein, n being equal to 2-3. It is shown that under the conditions of autolytic degradation both high molecular weight initiator based on trypsin and the trypsin-PVP (poly-N-vinyl pyrrolydone) covalent conjugates exhibit higher stability than initial trypsin. The method of circular dichroism was used for comparison of conformational properties of the modified trypsin forms. An increase of the rate of thermal inactivation was found to result from conformational changes occurring on modification of the enzyme.     

Chemical modification of arginine residues of rat liver S-adenosylhomocysteinase

Rat liver S-adenosylhomocysteinase (EC 3.3.1.1) is inactivated by phenylglyoxal following pseudo-first order kinetics. The dependence of the apparent first order rate constant for inactivation on the phenylglyoxal concentration shows that the inactivation is second order in reagent. This fact together with the reversibility of inactivation upon removal of excess reagent and the lack of reaction at residues other than arginine as revealed by amino acid analysis and incorporation of phenylglyoxal into the protein indicate that the inactivation is due to the modification of arginine residue. The substrate adenosine largely but not completely protects the enzyme against inactivation. Although the modification of two arginine residues/subunit is required for complete inactivation, the relationship between loss of enzyme activity and the number of arginine residues modified, and the comparison of the numbers of phenylglyoxal incorporated into the enzyme in the presence and absence of adenosine indicate that one residue which reacts very rapidly with the reagent compared with the other is critical for activity. Although the phenylglyoxal treatment does not result in alteration of the molecular size of the enzyme or dissociation of the bound NAD+, the intrinsic protein fluorescence is largely lost upon modification. The equilibrium binding study shows that the modified enzyme apparently fails to bind adenosine.