Chemical modifications of histidyl and tyrosyl residues of inorganic pyrophosphatase from Escherichia coli

Chemical modifications by photooxidation in the presence of rose bengal (RB) and with tetranitromethane (TNM) were carried out to elucidate the amino acid residues involved in the active site of inorganic pyrophosphatase (pyrophosphate phosphohydrolase) [EC 3.6.1.1] from Escherichia coli Q13. The photooxidation caused almost complete inactivation, which followed pseudo-first-order kinetics depending on pH and concentration of RB. The presence of Mg2+ or complex between Mg2+ and substrate or substrate analogues, imidodiphosphate and sodium methylenediphosphate, gave partial protection against the photoinactivation, whereas the substrate alone showed no protective effect. The enzyme was almost completely inactivated by chemical modification with TNM, depending upon the concentration of TNM. The amino acid analyses and enzyme activity measurements revealed that 2 histidyl residues among 5 photooxidized residues and 2 tyrosyl residues per subunit were essential for the enzyme activity. The circular dichroism (CD) spectra in the far ultraviolet region showed no significant alteration during these two modifications, indicating that the polypeptide chain backbone of the enzyme remained unaltered. However, the modifications altered considerably the CD bands in the near ultraviolet region and the fluorescence spectra, indicating that subtle change in conformation had occurred in the vicinity of the active site in the enzyme molecule. These results strongly suggest that histidyl and tyrosyl residues may be involved in the active site or be located in the vicinity of the active site and seem to participate in the mechanism of stability against heat inactivation.     

Effect of N-bromosuccinimide modification on dihydrofolate reductase from a methotrexate-resistant strain of Escherichia coli. Activity, spectrophotometric, fluorescence and circular dichroism studies.

When dihydrofolate reductase from a methotrexate-resistant strain of Escherichia coli B, MB 1428, is treated with approximately a 5 mol ratio of N-bromosuccinimide (NBS) to enzyme at pH 7.2 and assayed at the same pH, there is a 40% loss of activity due to the modification of 1 histidine residue and possibly 1 methionine residue before oxidation of tryptophan occurs. The initial modification is accompanied by a shift of the pH for maximal enzymatic activity from pH 7.2 to pH 5.5 Upon further treatment with N-bromosuccinimide, the activity is gradually reduced from 60 to 0% as tryptophan residues become oxidized. An NBS to enzyme mole ratio of approximately 20 results in 90% inactivation of the enzyme. When the enzyme is titrated with NBS in 6 M guanidine HCl, 5 mol of tryptophan react per mol of enzyme, a result in agreement with the total tryptophan content as determined by magnetic circular dichroism. The 40% NBS-inactivated sample posses full binding capacity for methotrexate and reduced triphosphopyridine nucleotide, and the Km values for dihydrofolate and TPNH are the same as for the native enzyme. After 90% inactivation, only half of the enzyme molecules bind methotrexate, and the dissociation constant for methotrexate is 40 nM as compared to 4 nM for native enzyme in solutions of 0.1 M ionic strength, pH 7.2 Also, TPNH is not bound as tightly to the modified enzyme-methotrexate complex as to the unmodified enzyme-methotrexate complex. Circular dichroism studies indicate the 90% NBS-inactivated enzyme has the same alpha helix content as the native enzyme but less beta structure, while the 40% inactivated enzyme is essentially the same as the native enzyme. Protection experiments were complicated by the fact that NBS reacts with the substrates and cofactors of the enzyme. Although protection of specific residues was not determined, it was clear that TPNH was partially protected from NBS reaction when bound to the enzyme, and the enzyme, and the enzyme was not inactivated by NBS until the TPNH had reacted.     

Cation-induced thermostability of yeast and Escherichia coli pyrophosphatases

Inorganic pyrophosphatases (PPiases) from both yeast and Escherichia coli were found to be stable against heat denaturation in the presence of Mg2+, as previously observed with the enzymes from thermophilic bacteria. No loss of activity was observed after 1 h of incubation at 50 degrees C and pHs between 6 and 9 in the yeast enzyme, and at 60 degrees C and pHs between 7.2 and 9.2 in the E. coli enzyme. Such an induced thermostability of the E. coli enzyme was detected when Mn2+, Co2+, Ca2+, Cd2+, and Zn2+ were added in place of Mg2+. On the other hand, the degree of induced thermostability of the yeast enzyme was dependent upon the divalent cations used, and Ni2+ and Cu2+ accelerated the heat inactivation. On adding the divalent cations, the difference spectra of the E. coli enzyme always showed negative peaks in the ultraviolet region, but those of the yeast enzyme changed again depending upon the divalent cations. The circular dichroism spectra in the near ultraviolet region of both enzymes greatly differed from each other, but both were not affected so much by adding the divalent cations unlike the thermophilic enzymes from Bacillus stearothermophilus and thermophilic bacterium PS-3. Yeast and E. coli PPiases did not cross-link with the anti-immunoglobulin G's from the thermophilic enzymes, but the thermophilic enzymes did with each other's antisera. The results in the present study indicated that the conformation of PPiase, in which the aromatic amino acid residues were buried in the interior of the protein molecule, was very important for the thermostability and also that the protein structures of PPiases from B. stearothermophilus and thermophilic bacterium PS-3 were very similar to each other, but were very different from those of the mesophilic enzymes.     

The chemical modification of the essential groups of beta-N-acetyl-D-glucosaminidase from Turbo cornutus Solander

The chemical modification of beta-N-acetyl-D-glucosaminidase (EC3.2.1.30) from Turbo cornutus Solander has been first studied. The results demonstrate that the sulfhydryl group of cysteine residues and the hydroxyl group of serine residues are not essential to the enzyme's function. The modification of indole group of tryptophan of the enzyme by N-bromosuccinimide (NBS) can lead to the complete inactivation, accompanying the absorption decreasing at 278 nm and the fluorescence intensity quenching at 335 nm, indicating that tryptophan is essential residue to the enzyme. The modification of amino group of lysine residue by formaldehyde and trinitrobenzenesulfonic acid also inactivates the enzyme completely. The results show that lysine and tryptophan are probably situated in the active site of the enzyme. The modification of the imidazole residue and carboxyl group leads to inactivate incompletely, indicating they are not the composing groups of the enzyme active center, and they are essential for maintaining the enzyme's conformation which is necessary for the catalytic activity of the enzyme.     

Circular dichroism of mammalian follitropins and the effects of treatment with N-bromosuccinimide

The circular dichroism of the tryptophan containing glycoprotein hormone, follitropin, displays bands in the near ultraviolet which are absent in homologous, tryptophan-free hormones. In the far ultraviolet, the dichroism is very similar to the other glycoprotein hormones with little or no indication of α-helix. The single tryptophan of follitropin is in a domain of the β subunit sequences of these hormones which is highly conserved from hormone to hormone. Without prior dissociation of the follitropin into subunits, no change is seen in circular dichroism, absorption at 280 nm, fluorescence emission or hormonal activity after treatment with N-bromosuccinimide. In contrast, these properties change when intact human lutropin is studied; its tryptophan residue is a position different than in follitropin. These results support the proposal that the domain containing the tryptophan in follitropin is in or near a region of subunit-subunit contact in the glycoprotein hormones.     

The modification of the lone tryptophan residue in human serum albumin by 2-hydroxy-5-nitrobenzyl bromide. Characterization of the modified protein and the binding of L-tryptophan and benzodiazepines to the tryptophan-modified albumin.

The possible function of the lone tryptophan residue of human serum albumin in the stereospecific binding site for indole and benzodiazepine compounds was investigated by chemical modification. This residue can be selectively modified with 2-hydroxy-5-nitrobenzyl bromide. The modification alters the conformation of the albumin only slightly, as revealed by circular dichroism, fluorescence, and ultraviolet absorption measurements. A decrease in the association constants of L-tryptophan and diazepam of about 30 - 50% and a decrease in the extrinsic Cotton effects of four benzodiazepine derivatives of about 10 - 15% were found as specific effects of the tryptophan modification. The tryptophan modification itself did not change the number of binding sites of diazepam and L-tryptophan. It is suggested that the lone tryptophan residue of human serum albumin is not directly involved in the specific binding site for indole and benzodiazepine compounds. However, the modification alters the properties of the binding site either by an incomplete refolding of the albumin after urea treatment, or a more selective allosteric effect of the modified tryptophan residue.     

Studies on the active site of rat glutathione S-transferase isoenzyme 4-4. Chemical modification by tetrachloro-1,4-benzoquinone and its glutathione conjugate

The active site of glutathione S-transferase isoenzyme 4-4, purified from rat liver, was studied by chemical modification. Tetrachloro-1,4-benzoquinone, a compound previously shown to inactivate glutathione S-transferases very efficiently by covalent binding in or close to the active site, completely prevented the alkylation of the enzyme by iodoacetamide, indicating that the reaction had taken place with cysteine residues. Both from radioactive labeling and spectral quantification experiments, evidence was obtained for the covalent binding of three benzoquinone molecules per subunit, i.e. equivalent to the number of cysteine residues present. This threefold binding was achieved with a fourfold molar excess of the benzoquinone, illustrating the high reactivity of this compound. Comparison of the number of amino acid residues modified by tetrachloro-1,4-benzoquinone with the decrease of catalytic activity revealed an almost complete inhibition after modification of one cysteine residue. Chemical modification studies with diethylpyrocarbonate indicated that all four histidine residues of the subunit are ethoxyformylated in an at least partially sequential manner. Modification of the second histidine residue resulted in complete loss of catalytic activity. Preincubation of the transferase with the glutathione conjugate of tetrachloro-1,4-benzoquinone resulted in 78% protection against this modification. However, glutathione itself hardly protected against the reaction with diethylpyrocarbonate. The intrinsic fluorescence properties of the enzyme were affected by covalent binding of tetrachloro-1,4-benzoquinone. The concentration dependency of the fluorescence quenching is strongly correlated with the inactivation of the enzyme, indicating that covalent binding of the benzoquinone occurs in the vicinity of at least one tryptophan residue. Finally, the binding of bilirubin, as measured by means of circular dichroism, was inhibited by preincubation of the enzyme with tetrachloro-1,4-benzoquinone in a manner which strongly correlated with the loss of enzymatic activity, the protection against inactivation by diethylpyrocarbonate, and the fluorescence quenching. All processes showed a 70-80% decrease after incubation of the enzyme with an equimolar amount of the benzoquinone. Thus, evidence is presented for the presence of a cysteine, a histidine and a tryptophan residue in, or in the vicinity of, the active site of the glutathione S-transferase 4 subunit.     

葡萄糖淀粉酶色氨酸残基及底物结合位点的研究

 本文用N-溴代琥珀酰亚胺(NBS)对葡萄糖淀粉酶进行特异性修饰,当酶分子表面有3个色氨酸残基被修饰后,酶活力完全丧失。用邹氏图解法测得酶活性中心有一个色氨酸残基是必需的。如果在酶液中加入不同的底物再用NBS氧化,用荧光发射和荧光猝灭光谱检测表明,底物对酶分子有不同程度的保护作用。在被测试的三种底物中,这种保护能力依为糊精>淀粉>麦芽糖。     

Conformational changes of papain induced on interaction with thiol proteinase inhibitors from newborn rat epidermis

The conformational changes of the papain molecular on interaction with two thiol proteinase inhibitors (TPI(1) and TPI(2] from newborn rat epidermis were studied by measuring circular dichroism (CD), the difference absorption spectrum, and the fluorescence spectrum due to tryptophan residues in papain. The far-ultraviolet CD band of papain between 210 and 230 nm was distinctly reduced on interaction with both inhibitors. Also, the near-ultraviolet CD spectrum of TPI(1)-bound papain changed between 285 and 320 nm as well as that of the TPI(2)-bound enzyme. The difference absorption spectrum for TPI(1)-bound papain exhibited two distinct peaks at 276.5 and 282 nm, indicating perturbation of aromatic amino acid residues. The fluorescence intensity of papain was significantly decreased on interaction with both inhibitors, which showed pH-dependency on an ionizable group, with pK values of 8.5 and 7.9 for TPI(1) and TPI(2), respectively. The complex formation of papain with both inhibitors caused a reduction of the susceptibility of a tryptophan residue, probably tryptophan-177, to chemical modification with N-bromosuccinimide. These results suggest that the active site involving histidine-159 in the papain molecule was much influenced by the alteration of the microenvironment of tryptophan-177 as a part of the interaction site for these two thiol proteinase inhibitors.     

米曲霉GX0011β-果糖基转移酶的化学修饰及活性中心研究

用化学修饰法及其修饰动力学对米曲霉GX0011β-果糖基转移酶的活性中心结构进行了研究。结果表明：NBS、PMSF、EDC能显著抑制酶的活性,底物对这些抑制有明显的保护作用,且残留酶活与修饰剂的浓度相关,抑制均符合拟一级动力学规律,进一步动力学分析,初步认定该酶活性中心包括至少一个丝氨酸（或苏氨酸）、一个色氨酸和一个天冬氨酸(或谷氨酸)残基。pCMB、TNBS能显著抑制酶的活性,但底物对抑制无明显保护作用,推断半胱氨酸和赖氨酸残基可能与维系酶活性中心构象有关,但不是酶活性中心基团。DEPC、AA和NAI对酶的活性抑制作用不明显,排除了组氨酸、精氨酸和酪氨酸残基是该酶活性中心必需基团的可能。     

p-Chloromercuribenzoate-induced inactivation and partial unfolding of porcine heart lactate dehydrogenase

Purified porcine heart lactate dehydrogenase was inactivated and partially unfolded with p-chloromercuribenzoate (pCMB). With the increase of pCMB/enzyme ratio the enzyme was gradually inhibited till almost completely inactivated at the pCMB/enzyme ratio of 20 : 1. Native polyacrylamide gel electrophoresis showed that with the increase of pCMB/enzyme ratio the bands of native enzyme decreased till completely vanished. Meanwhile inactive multiple bands emerged and became thicker, which implied that lactate dehydrogenase became loose. The conformational changes of the enzyme molecule modified with pCMB were followed using fluorescence emission, ultraviolet difference, and circular dichroism (CD) spectra. Increasing pCMB concentration resulted in the decrease of fluorescence emission intensity. The ultraviolet difference spectra of the enzyme modified with pCMB exhibited an increasing absorbance in the vicinity of 240 nm with the increasing concentration of the inhibitor. The changes of the fluorescence and ultraviolet difference spectra reflected the conformational changes of the enzyme. The CD spectrum changes of the enzyme showed that its secondary structure changed as well. These results suggest that pCMB not only inhibits this enzyme but also influences its conformation (partial unfolding).     

Divalent cation-induced changes in conformation of protein kinase C

Using physical techniques, circular dichroism and intrinsic and extrinsic fluorescence, the binding of divalent cations to soluble protein kinase C and their effects on protein conformation were analyzed. The enzyme copurifies with a significant concentration of endogenous Ca2+ as measured by atomic absorption spectrophotometry, however, this Ca2+ was insufficient to support enzyme activity. Intrinsic tryptophan fluorescence quenching occurred upon addition to the soluble enzyme of the divalent cations, Zn2+, Mg2+, Ca2+ or Mn2+, which was irreversible and unaffected by monovalent cations (0.5 M NaCl). Far ultraviolet (200-250 nm) circular dichroism spectra provided estimations of secondary structure and demonstrated that the purified enzyme is rich in alpha-helices (42%) suggesting a rather rigid structure. At Ca2+ or Mg2+ concentrations similar to those used for fluorescence quenching, the enzyme undergoes a conformational transition (42-24% alpha-helix, 31-54% random structures) with no significant change in beta-sheet structures (22-26%). Maximal effects on 1 microM enzyme were obtained at 200 microM Ca2+ or 100 microM Mg2+, the divalent cation binding having a higher affinity for Mg2+ than for Ca2+. The Ca2(+)-induced transition was time-dependent, while Mg2+ effects were immediate. In addition, there was no observed energy transfer for protein kinase C with the fluorescent Ca2(+)-binding site probe, terbium(III). This study suggests that divalent cation-induced changes in soluble protein kinase C structure may be an important step in in vitro analyses that has not yet been detected by standard biochemical enzymatic assays.     

The environments of Trp-248 and Trp-330 in tryptophan indole-lyase from Escherichia coli

The two tryptophan residues, Trp-248 and Trp-330, in tryptophan indole-lyase (tryptophanase) from E. coli have been separately mutated to phenylalanine using site-directed mutagenesis. Both single tryptophan mutant enzymes have full catalytic activity, but exhibit different fluorescence and near-UV circular dichroism spectra. These results indicate that Trp-330 is more deeply buried than is Trp-248, and is in a more asymmetric environment. Neither residue reacts with N-bromosuccinimide (NBS), although tryptophan indole-lyase is inactivated by NBS. These results demonstrate that the tryptophan residues in tryptophan indole-lyase are not catalytically essential.     

The number and role of histidine residues in the active site of guanyloribonuclease Sa

The number and role of histidine residues in the active site of extracellular guanyloribonuclease Sa produced by Streptomyces aureofaciens (RNAase Sa) were studied via chemical modification by ethoxyformic anhydride by means of circular dichroism measurements. It was shown that only one of two histidines of RNAase Sa is situated in the active site of the enzyme. Ethoxyformylation of RNAase Sa in the presence of Guo-3'-P, Guo-5'-P and dGuo-5-P, all of them being competitive inhibitors of the enzyme, supported the assumption that an essential histidine residue is bound to the phosphate group in the position 3' of the ribose ring. The circular dichroism measurements of native and modified RNAase Sa and of its complex with Guo-3'-P showed that the modification of the essential histidine residue resulted in alteration of binding of RNAase Sa to Guo-3'-P; histidine thus may play a key role in the formation of such a complex.     

Role of tryptophan in the spectral and catalytic properties of the copper enzyme, galactose oxidase.

Previous results indicate that a tryptophan residue(s) may interact with the sugar substrate and Cu(II) atom of galactose oxidase (Ettinger, M. J., and Kosman, D. J. (1974), Biochemistry 13, 1248). We now show that N-bromosuccinimide (NBS) reduces enzymatic activity to 2% as two tryptophans are oxidized; only four residues are easily oxidized in the holoenzyme. An enzymatic activity vs. number of residues oxidized profile suggests that this inactivation is probably associated with only one of the first 2 residues oxidized. There is no evidence for chain cleavage or modification of amino acids other than tryptophan. While substrate protection is not afforded by the sugar substrate, the activity-related tryptophan is placed within the active-site locus by spectral evidence. NBS oxidation of two tryptophans results in a marked diminution of the large copper optical-activity transition at 314 nm. Under some reaction conditions, a doubling of ellipticity in the 600-nm region of copper CD is also observed. The effects of the NBS oxidation on the CD spectra of galactose oxidase permit the assignment of the 314-nm CD band to a charge-transfer transition and the 229-nm extremum to a specific tryptophan contribution. The AZZ parameter from electron spin resonance spectra is also markedly reduced by the NBS oxidation. Moreover, while cyanide binds to the native enzyme without reducing the Cu(II) atom, cyanide rapidly reduces the Cu(II) atom to Cu(I) in the NBS-oxidized enzyme. These CD and ESR results are taken to suggest that one aspect of the inactivation by NBS oxidation may be a conversion of the pseudosquare planar copper complex in the native enzyme to a more distorted, towards tetrahedral, complex in the inactivated enzyme. Since the inactivation can be accomplished without affecting binding of the sugar substrate, tryptophan oxidation must affect catalysis per se.     

Circular dichroism and absorbance properties of nitrotyrosyl chromophores in staphylococcal nuclease and in a model diketopiperazine.

An active derivative of staphylococcal nuclease, in which only tyrosine residue 115 has been nitrated with use of tetranitromethane, has been characterized using absorbance, circular dichroism, and fluorescence spectroscopy. The results show that nitrotyrosine-115 nuclease is indistinguishable from native nuclease with regard to the average secondary structure of the folded polypeptide chain, the susceptibility of the enzyme to heat denaturation, and the local tertiary structure around tryptophan residue 140. Inasmuch as optical properties of nitrotyrosine-115 nuclease from 300 to 500 nm can be unambiguously assigned to nitrotyrosine residue 115 in the active site region, this modified enzyme presents a good model system for studying the circular dichroism properties of this aromatic amino acid in a protein. The spectral properties of nitrotyrosine-115 nuclease have been compared to those of the model compounds, cyclo-(-Gly-Tyr(3NO2)-) and Tyr(3NO2). Circular dichroism spectral changes in nitrotyrosine-115 nuclease due to the binding of deoxythymidine 3',5'-diphosphate and Ca-2+ have been compared to the corresponding nitrotyrosyl-115 absorption spectral changes. This comparison shows that the circular dichroism difference spectrum exhibits an over-all change in the intensity of the observed Cotton effects, whereas the absorption difference spectrum exhibits a blue shift. This finding supports the suggestion that perturbations of aromatic amino acid chromophores in proteins due to ligand binding result in red or blue shifts in absorption difference spectra, but in over-all changes of intensity in circular dichroism difference spectra.     

Tryptophan residues of saccharifying alpha-amylase from Bacillus subtilis. A kinetic discrimination of states of tryptophan residues using N-bromosuccinimide.

Four tryptophan residues of saccharifying alpha-amylase from B. subtilis out of eleven in total are reactive towards N-bromosuccinimide (NBS), suggesting that they are on the surface of the enzyme. This is consistent with the results of solvent perturbation difference spectrophotometry with ethylene glycol. One of four tryptophan residues was clearly distinguished from the other three in reactivity with NBS by the stopped-flow method. This most reactive tryptophan residue was not protected from modification by substrates of analogs, indicating that the tryptophan is not located in the substrate binding site. One of the other three tryptophan residues, probably the second most reactive one, is considered to be related in some way to the glycosyl transfer in the reaction of the enzyme with maltose as a substrate.     

Binary and ternary complexes of dihydrofolate reductase with substrates, coenzymes and inhibitors. Circular dichroic and magnetic circular dichroic studies.

Interaction of several representative folate, quinazoline and pyridine nucleotide derivatives with dihydrofolate reductase from amethopterin-resistant Lactobacillus casei induces dramatic changes in its circular dichroic spectral properties. The binding of dihydrofolate induces a large extrinsic Cotton effect at 295 nm ([theta] = 113 800 deg . cm2 . dm-1). The generation of this band by dihydrofolate is strictly dependent on complex formation with a single substrate binding site and a KD = 7 . 10(-6) M. The other binary complexes examined include the enzyme . NADPH, enzyme . amethopterin, enzyme . folate, and enzyme . methasquin. All such complexes differ in spectral detail, the negative ellipticity at 330 nm being characteristic of the "folate site" complexes. The circular dichroic spectrum of the ternary complex of reductase . NADPH . methotrexate shows a positive symmetrical band centered at 360 nm ([theta] - 32 000 deg . cm2 . dm-1). Since both of the corresponding binary complexes exhibit negative bands in this region, this induced band represents a unique molecular property of the ternary complex. Chemical modification of a single tryptophan residue of the enzyme, as determined from magnetic circular dichroism spectra, results in a complete loss in the ability to bind either dihydrofolate or NADPH.     

Inhibition of myosin ATPase by metal fluoride complexes

Magnesium (Mg2+) is the physiological divalent cation stabilizing nucleotide or nucleotide analog in the active site of myosin subfragment 1 (S1). In the presence of fluoride, Mg2+ and MgADP form a complex that traps the active site of S1 and inhibits myosin ATPase. The ATPase inactivation rate of the magnesium trapped S1 is comparable but smaller than the other known gamma-phosphate analogs at 1.2 M-1 s-1 with 1 mM MgCl2. The observed molar ratio of Mg/S1 in this complex of 1.58 suggests that magnesium occupies the gamma-phosphate position in the ATP binding site of S1 (S1-MgADP-MgFx). The stability of S1-MgADP-MgFx at 4 degrees C was studied by EDTA chase experiments but decomposition was not observed. However, removal of excess fluoride causes full recovery of the K+-EDTA ATPase activity indicating that free fluoride is necessary for maintaining a stable trap and suggesting that the magnesium fluoride complex is bonded to the bridging oxygen of beta-phosphate more loosely than the other known phosphate analogs. The structure of S1 in S1-MgADP-MgFx was studied with near ultraviolet circular dichroism, total tryptophan fluorescence, and tryptophan residue 510 quenching measurements. These data suggest that S1-MgADP-MgFx resembles the M**.ADP.Pi steady-state intermediate of myosin ATPase. Gallium fluoride was found to compete with MgFx for the gamma-phosphate site in S1-MgADP-MgFx. The ionic radius and coordination geometry of magnesium, gallium and other known gamma-phosphate analogs were compared and identified as important in determining which myosin ATPase intermediate the analog mimics.     

诺卡氏菌形放线菌β-甘露聚糖酶的化学修饰及活性中心的研究

分别用 PCMB、NEM、N- AI、NBS等对诺卡氏菌形放线菌β- D-甘露聚糖酶进行化学修饰 ,证明蛋白上的巯基、酪氨酸残基及色氨酸残基是维持酶活性的必需基团 .在加入少量底物后 ,β- D-甘露聚糖酶的最大荧光发射峰从天然状态下的 336nm处蓝移至 332 nm,且峰强度有所增大 .这表明其色氨酸残基隐藏在蛋白内部的疏水区域 .通过对该酶圆二色性扫描光谱的分析 ,表明蛋白内部有二硫键的存在 ;通过巯基乙醇化学修饰的研究 ,表明二硫键是影响该酶热稳定性的一个重要因素 .在蛋白的各种二级结构中 ,α-螺旋、β-折叠、β-转角、自由卷曲的比例分别为 1 6.6%、2 5.4%、2 0 .5%和 37.5% .