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1.
Effect of chaotropic agents on the structure-function of recombinant acylpeptide hydrolase 总被引:1,自引:0,他引:1
Acylpeptide hydrolase, a new class the serine-type peptidase, belongs to the , hydrolase group of proteins. The tetrameric enzyme showed varying degree of stability in the presence of 1–8 M urea. The enzyme displayed about 15% of its original activity when treated with 8 M urea for 1 h at 25°C. Complete recovery of the enzyme activity was observed on dialysis or dilution (50-fold) of the denatured enzyme. However, complete abolition of the enzyme activity was observed in the presence of 1 M GnHCl. Dialysis of the 1 M GnHCl-treated enzyme resulted in 15–20% recovery of the enzyme activity. The fluorescence emission spectra of the native enzyme at 337 nm showed a red shift up to 16 nm in 8 M urea and 18 nm in the presence of 4 M GnHCl. Native enzyme during far-UV circular dichroism spectroscopy exhibited predominantly -sheet structure. The enzyme lost its secondary structure at urea concentrations of 2 M and higher, whereas the tertiary structure was minimally perturbed below 4 M urea. However, in 1 M GnHCl the enzyme lost both its secondary and tertiary structures and the enzyme was found to dissociate into monomers of 70 kDa. Both monomeric and dimeric species were observed after 24-h dialysis of the enzyme denatured with GnHCl indicating the reassociation process. Both monomer and dimers forms recovered after dialysis were active. 相似文献
2.
Perrier J Giardina T Durand A Puigserver A 《Biology of the cell / under the auspices of the European Cell Biology Organization》2002,94(1):45-54
The specific effects of N-acetyl-L-methionine on acylase I activity and of both N-acetyl-L-alanyl-L-alanine and N-acetyl-L-methionyl-L-alanine on N-acylpeptide hydrolase activity were investigated in primary rat hepatocyte cultures. Each of the above two substrates is known to be much more rapidly hydrolyzed than other derivatives of the same type under optimum enzyme assay conditions. After a two-day incubation of the substrates in the presence of primary rat hepatocyte cultures, the N-acetylaminoacid was found to specifically induce an increase in the acylase I activity, whereas the two N-acetylated peptides increased the acylpeptide hydrolase activity in the soluble 100,000 x g fraction from the culture medium. No change in any of the enzyme activities could be detected during the same period of time when the medium was not supplemented with N-acetylated substrates. In addition, the acylase I activity showed a dose dependent response when the N-acetyl-L-methionine concentration increased from 10 fold to 50 fold. It is therefore suggested that the efficient hydrolysis of each type of substrate that occured in the 48 h hepatocyte cell cultures was due to the increase observed in the overall activity of the corresponding enzymes. The ratio of acylpeptide hydrolase to that of acylase I increased considerably when the medium was supplemented with N-acetylpeptides, and decreased in the presence of the N-acetylaminoacid. 相似文献
3.
Andrea Scaloni Paolo Ingallinella Annapaola Andolfo Wanda Jones Gennaro Marino James M. Manning 《Journal of Protein Chemistry》1999,18(3):349-360
The complete primary structure of human erythrocyte acylpeptide hydrolase has been determined by using a combination of different mass spectrometric procedures and sequencing techniques. These data allowed us to correct the incomplete nucleotide sequence of the DNF15S2 locus on the short arm of human chromosome 3 at region 21, coding for the enzyme. The protein consists of 732 amino acid residues and is acetylated at the N-terminus. Alkylation experiments on the native enzyme demonstrated that all 17 cysteine residues present in the polypeptide chain are in reduced form. Multiple sequence alignment did not reveal striking similarity with proteases of known tertiary structure with the exception of members of the serine oligopeptidase family. Limited proteolysis experiments generated a C-terminal portion, containing all the catalytic triad elements responsible for proteolytic activity, and an N-terminal domain of unknown function, both still strongly associated in a completely active nicked form. The site of tryptic hydrolysis was identified as Arg193. The secondary structural organization of the protease domain of the enzyme is consistent with the / hydrolase fold. 相似文献
4.
Effect of limited proteolysis on the stability and enzymatic activity of human placental S-adenosylhomocysteine hydrolase.
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H. Huang C. S. Yuan R. T. Borchardt 《Protein science : a publication of the Protein Society》1997,6(7):1482-1490
Human placental S-adenosylhomocysteine (AdoHcy) hydrolase was subjected to limited papain digestion. The multiple cleavage sites in the enzyme were identified to be Lys94-Ala95, Tyr100-Ala101, Glu243-Ile244, Met367-Ala368, Gln369-Ile370, and Gly382-Val383. Despite multiple cleavage sites in the backbone of the protein, the digested enzyme was able to maintain its quaternary structure and retain its full catalytic activity. The enzyme activity of the partially digested AdoHcy hydrolase was essentially identical to that of the native enzyme at several pH values. The thermal stabilities of the native and partially digested enzymes were only slightly different at all temperatures tested. The stability of both native and partially digested enzymes were examined in guanidine hydrochloride and equilibrium unfolding transitions were monitored by CD spectroscopy and tryptophan fluorescence spectroscopy. The results of these experiments can be summarized as follows: (1) CD spectroscopic analysis showed that the overall secondary and tertiary structures of the partially digested enzyme are essentially identical with those of the native enzyme; and (2) tryptophan fluorescence spectroscopic analysis indicated that there are small differences in the environments of surface-exposed tryptophan residues between the partially digested enzyme and the native enzyme under unfolding conditions. The differences in the free energy of unfolding, delta(delta Gu) [delta Gu(native)-delta Gu(digested)], is approximately 1.3 kcal/mol. When NAD+ was removed from the partially digested enzyme, the secondary and tertiary structures of the apo form of the digested AdoHcy hydrolase were completely lost and the enzymatic activity could not be recovered by incubation with excess NAD+. These results suggest that AdoHcy hydrolase exists as a very compact enzyme with extensive intramolecular bonding, which contributes significantly to the overall global protein stabilization. Identification of the surface-exposed peptide bonds, which are susceptible to papain digestion, has provided some constraints on the spatial orientations of subunits of the enzyme. This information, in turn, has provided supplemental data for X-ray crystallographic studies currently ongoing in our laboratories. 相似文献
5.
Zhang HF Zheng BS Peng Y Lou ZY Feng Y Rao ZH 《Acta biochimica et biophysica Sinica》2005,37(9):613-617
Acylpeptide hydrolase (APH) catalyzes the N-terminal hydrolysis of N^α-acylpeptides to release N^α-acylated amino acids. The crystal structure of recombinant APH from the thermophilic archaeon Aeropyrum pernix K1 (apAPH) was reported recently to be at a resolution of 2.1 A using X-ray diffraction. A truncated mutant of apAPH that lacks the first short α-helix at the N-terminal, apAPH-△(1-21), was cloned, expressed, characterized and crystallized. Data from biochemical experiments indicate that the optimum temperature of apAPH is decreased by 15℃ with the deletion of the N-terminal α-helix. However, the enzyme activity at the optimal temperature does not change. It suggests that this N-terminal α-helix is essential for thermostability. Here, the crystal structure of apAPH-△(1-21) has been determined by molecular replacement to 2.5 A. A comparison between the two structures suggests a difference in thermostability, and it can be concluded that by adding or deleting a linking structure (located over different domains), the stability or even the activity of an enzyme can be modified. 相似文献
6.
Cloning, sequencing and further characterization of acylpeptide hydrolase from porcine intestinal mucosa. 总被引:3,自引:0,他引:3
V Raphel T Giardina L Guevel J Perrier L Dupuis X J Guo A Puigserver 《Biochimica et biophysica acta》1999,1432(2):371-381
Acylpeptide hydrolase was purified to homogeneity from porcine intestinal mucosa using a seven-step procedure including ammonium sulfate precipitation, gel filtration as well as anion exchange and affinity chromatography. The specific activity of the enzyme reached 105000 nmol/mg protein per min and the purification was as high as 5500-fold. This tetrameric enzyme is composed of four apparently identical subunits, the molecular mass of which was estimated to be 75 kDa, based on the results of amino acid analysis and gel electrophoresis performed under denaturing conditions. It is likely that the NH(2)-terminal residue may be acetylated, while serine was found to be the COOH-terminal residue. The hydrolytic activity of the enzyme toward N-acetyl-L-alanine p-nitroanilide at the optimum pH value was increased twofold in the presence of the chloride anion. The K(m) value calculated from the kinetics of the hydrolysis of acetylalanyl peptides was found to be 0.7+/-0.1 mM, whereas the V(max) values decreased from 200 to 50 nmol/min per microgram of enzyme, depending on the peptidic chain lengths. The V(max) value of the synthetic substrate (250 nmol/min per microgram of enzyme) was 25-500% higher than those of the acetylalanyl peptides, depending on the peptide chain length, although the enzyme affinity was slightly lower (1.8 mM as compared with 0.7 mM). In line with data on other animal species and on various tissues, the enzyme seemed likely to be a serine protease, since it was readily inhibited by diisopropyl fluorophosphate and diethyl pyrocarbonate. A 2377-nucleotide long cDNA coding for the enzyme was isolated from pig small intestine. The deduced amino acid sequence consisted of 731 residues and showed a single different amino acid with that of the porcine liver APH, except the N-terminal amino acid which is still probably lacking. 相似文献
7.
Elena Efremenko Ilya Lyagin Yulia Votchitseva Maria Sirotkina Sergey Varfolomeyev 《Biocatalysis and Biotransformation》2013,31(1):103-108
The catalytic and physical–chemical properties of organophosphorus hydrolase (OPH) modified by the addition of an N-terminal dodecahistidine tag (His12-OPH) have been investigated. Introduction of the His12-tag caused a 30- and 74-fold increase in catalytic efficiency of the enzyme with parathion and methyl parathion, respectively, compared to OPH. Concurrently, the His12-OPH had a more alkaline pH-optimum and extended temperature range than OPH and OPH modified with a hexahistidine tag. A study of His12-OPH thermostability showed that the enzyme had a tendency to oligomerise. This resulted in a decrease in the enzymatic activity of His12-OPH at temperatures <50°C, but provided the enzyme with much higher thermostability at temperatures >50°C, compared to OPH. 相似文献
8.
Sujata Sharma Sanket KaushikMau Sinha Gajraj Singh KushwahaAvinash Singh Juhi SikarwarAnshul Chaudhary Akshita GuptaPunit Kaur Tej P. Singh 《Biochimica et Biophysica Acta - Proteins and Proteomics》2014,1844(7):1279-1288
Peptidyl-tRNA hydrolase is an essential enzyme which acts as one of the rescue factors of the stalled ribosomes. It is an esterase that hydrolyzes the ester bond in the peptidyl-tRNA molecules, which are products of ribosome stalling. This enzyme is required for rapid clearing of the peptidyl-tRNAs, the accumulation of which in the cell leads to cell death. Over the recent years, it has been heralded as an attractive drug target for antimicrobial therapeutics. Two distinct classes of peptidyl-tRNA hydrolase, Pth and Pth2, have been identified in nature. This review gives an overview of the structural and functional aspects of Pth, along with its sequence and structural comparison among various species of bacteria. While the mode of binding of the substrate to Pth and the mechanism of hydrolysis are still speculated upon, the structure-based drug design using this protein as the target is still largely unexplored. This review focuses on the structural features of Pth, giving a direction to structure-based drug design on this protein. 相似文献
9.
Kamatari YO Dobson CM Konno T 《Protein science : a publication of the Protein Society》2003,12(4):717-724
It has been established in a number of studies that the alkaline-denatured state of pepsin (the I(P) state) is composed of a compact C-terminal lobe and a largely unstructured N-terminal lobe. In the present study, we have investigated the residual structure in the I(P) state in more detail, using limited proteolysis to isolate and characterize a tightly folded core region from this partially denatured pepsin. The isolated core region corresponds to the 141 C-terminal residues of the pepsin molecule, which in the fully native state forms one of the two lobes of the structure. A comparative study using NMR and CD spectroscopy has revealed, however, that the N-terminal lobe contributes a substantial amount of additional residual structure to the I(P) state of pepsin. CD spectra indicate in addition that significant nonnative alpha-helical structure is present in the C-terminal lobe of the structure when the N-terminal lobe of pepsin is either unfolded or removed by proteolysis. This study demonstrates that the structure of pepsin in the I(P) state is significantly more complex than that of a fully folded C-terminal lobe connected to an unstructured N-terminal lobe. 相似文献
10.
John Mosior Ronnie Bourland Shivatheja Soma Carl Nathan James Sacchettini 《Protein science : a publication of the Protein Society》2020,29(3):744-757
The amidinourea 8918 was recently reported to inhibit the type II phosphopantetheinyl transferase (PPTase) of Mycobacterium tuberculosis (Mtb), PptT, a potential drug‐target that activates synthases and synthetases involved in cell wall biosynthesis and secondary metabolism. Surprisingly, high‐level resistance to 8918 occurred in Mtb harboring mutations within the gene adjacent to pptT, rv2795c, highlighting the role of the encoded protein as a potentiator of the bactericidal action of the amidinourea. Those studies revealed that Rv2795c (PptH) is a phosphopantetheinyl (PpT) hydrolase, possessing activity antagonistic with respect to PptT. We have solved the crystal structure of Mtb's phosphopantetheinyl hydrolase, making it the first phosphopantetheinyl (carrier protein) hydrolase structurally characterized. The 2.5 Å structure revealed the hydrolases' four‐layer (α/β/β/α) sandwich fold featuring a Mn‐Fe binuclear center within the active site. A structural similarity search confirmed that PptH most closely resembles previously characterized metallophosphoesterases (MPEs), particularly within the vicinity of the active site, suggesting that it may utilize a similar catalytic mechanism. In addition, analysis of the structure has allowed for the rationalization of the previously reported PptH mutations associated with 8918‐resistance. Notably, differences in the sequences and predicted structural characteristics of the PpT hydrolases PptH of Mtb and E. coli's acyl carrier protein hydrolase (AcpH) indicate that the two enzymes evolved convergently and therefore are representative of two distinct PpT hydrolase families. 相似文献
11.
【目的】随着抗生素生长促进剂(AGPs)在动物饲料中逐步禁止使用,AGPs替代物的研究成为热点。由于胆盐水解酶(BSH)在脂类代谢中的关键作用,成为AGPs替代物研究的一个重要方向。在原核表达和纯化的基础上鉴定鸡源和猪源乳杆菌BSH在酶学性质方面的差异性。【方法】分别对鸡源胆盐水解酶(BSHc)和猪源胆盐水解酶(BSHp)基因进行原核表达和蛋白纯化,通过测定对6种甘氨结合胆盐和牛磺结合胆盐的水解效率获得两种酶的酶学动力学性质,进而测定了温度、pH和金属离子对酶活力的影响。【结果】BSHc和BSHp对甘氨结合胆盐的水解效率高于牛磺结合胆盐,BSHc对甘氨结合胆盐的水解效率较BSHp稍高;BSHc和BSHp的最适酶解温度分别为45°C和42°C;BSHc和BSHp的最适反应pH分别为6.0和5.4;含有Cu~(2+)、Fe~(3+)、Mn~(2+)和Zn~(2+)的金属盐对BSHc和BSHp的酶活力均具有不同程度的抑制作用,特别是Cu~(2+)和Fe~(3+)抑制作用比较强;含有Na~+、K~+、Mg~(2+)和Ca2+的金属盐对BSHc和BSHp酶活力的抑制作用相对较弱或无抑制作用,但KIO3对BSHc和BSHp酶活力具有强抑制作用,KI和CaCl_2对BSHp酶活力也具有较强的抑制作用。【结论】原核表达和纯化的BSHc和BSHp对甘氨结合胆盐的水解效率高于牛磺结合胆盐,BSHc的最适酶解温度和pH稍高于BSHp,Cu~(2+)、Fe~(3+)、Mn~(2+)和Zn~(2+)等金属离子对BSHc和BSHp酶活力具有明显抑制作用,试验结果为鉴定BSH抑制物进而研制AGPs替代物奠定了基础。 相似文献
12.
Bile salt hydrolase (BSH), the enzyme deconjugating bile potentially plays an important role in reduction of blood cholesterol level. BSH enzymes from various sources differ in characteristics, substrates preference and specific catalytic activity. In this study, two BSH enzymes (BSH1 and BSH2) from Lactobacillus salivarius were heterologously expressed and purified. Both of them were characterized as homotetramer according to their molecular weight from size exclusion chromatograph. BSH1 showed a broad pH optimum over the range from 5.5 to 7.0, while a narrower range of pH optimum from 5.5 to 6.0 for BSH2 was detected. The enzymatic kinetics of the purified BSH1 and BSH2 have demonstrated BSH enzymes from bacteria were allosteric enzymes, and have also revealed their striking differences in positive cooperativity, catalytic efficiency and substrate preference for the first time. In contrast to the enzymatic reactions of BSH in the absence of dithiothreitol, the kinetics curves of BSH1 and BSH2 were similar to hyperbolic forms of Michaelis–Menten kinetics in the presence of dithiothreitol. 相似文献
13.
V. N. R. Kartha N. Jayanthi Bai Thomas George S. Krishnamurthy 《Journal of biosciences》1979,1(2):159-168
An enzyme catalysing the hydrolysis ofa-tocopheryl acetate was characterised in chicken liver. The enzyme was localised in the microsomes, had an optimum pH 8.6
and aK
m value of 0.5 mM. The enzyme did not hydrolyse retinyl acetate, cholesteryl acetate and ethyl acetate, thus indicating a high
degree of specificity.a-Tocopheryl acetate hydrolase required bile salts as a specific co-factor. The results suggested a role for this enzyme in
the absorption of vitamin E. 相似文献
14.
Residue-specific chemical modification of amino acid residues of the microsomal epoxide hydrolase (mEH) from Rhodosporidium toruloides UOFS Y-0471 revealed that the enzyme is inactivated through modification of Asp/Glu and His residues, as well as through modification of Ser. Since Asp acts as the nucleophile, and Asp/Glu and His serve as charge relay partners in the catalytic triad of microsomal and soluble epoxide hydrolases during epoxide hydrolysis, inactivation of the enzyme by modification of the Asp/Glu and His residues agrees with the established reaction mechanism of these enzymes. However, the inactivation of the enzyme through modification of Ser residues is unexpected, suggesting that a Ser in the catalytic site is indispensable for substrate binding by analogy of the role of Ser residues in the related L-2-haloacid dehalogenases, as well as the ATPase and phosphatase enzymes. Co2+, Hg2+, Ag+, Mg2+ and Ca2+ inhibited enzyme activity and EDTA increased enzyme activity. The activation energy for inactivation of the enzyme was 167 kJ mol–1. Kinetic constants for the enzyme could not be determined since unusual behaviour was displayed during hydrolysis of 1,2-epoxyoctane by the purified enzyme. Enantioselectivity w as strongly dependent on substrate concentration. When the substrate was added in concentrations ensuring two-phase conditions, the enantioselectivity was greatly enhanced. On the basis of these results, it is proposed that this enzyme acts at an interface, analogous to lipases. 相似文献
15.
Hisataka Awata Fumio Endo Akito Tanoue Akito Kitano Yoshikuni Nakano Ichiro Matsuda 《生物化学与生物物理学报:疾病的分子基础》1994,1226(2):168-172
Fumarylacetoacetate hydrolase (FAH) is a metabolic enzyme functioning at the last steo of tyrosine catabolism. Deficiency in this enzyme activity is associated with tyrosinemia type I, characterized by hypertyrosinemia, liver dysfunction, renal tubular dysfunction, liver cirrhosis, and hepatic tumors. We isolated from a human gene library a chromosomal gene related to FAH. The human FAH gene is 30 kilobases long and is split into 14 exons. All of the splice donor and acceptor sites conform to the GT/AG rule. We also analyzed findings in a patient with tyrosinemia type I with respect to the mutation responsible for detects in the enzyme. A nucleotide change from T to G was found in the exon 2 of the gene and this change was accompanied by an amino acid substitution (Phe62Cys). Transfection and expression analysis of the cDNA is cultured BMT-10 cells with the nucleotide substitution demonstrated that the substitution was indeed responsible for the decreased activity of the enzyme in the patient. These results confirmed that the T to G mutation was one of the causes of tyrosinemia type I. Structure of the FAH gene and tests for expression of the mutant FAH will facilitate further understanding of various aspects of FAH. 相似文献
16.
Glenn S. Maitz Edward M. Haas Peter A. Castric 《Biochimica et Biophysica Acta (BBA)/General Subjects》1982,714(3):486-491
Allophanate hydrolase was purified to homogeneity from extracts of Chlamydomonas reinhardii grown phototrophically using urea as sole source of nitrogen. The following sequence of steps comprised the purification procedure: (1) protamine sulfate precipitation; (2) ammonium sulfate fractionation; (3) poly(ethylene glycol) fractionation; (4) batch-wise DEAE-cellulose adsorption; (5) Sepharose 6-B gel filtration; (6) hydroxyapatite chromatography. This procedure yielded an allophanate hydrolase preparation which was homogenous as judged by polyacrylamide gel electrophoresis. The molecular weight, as determined by gradient polyacrylamide electrophoresis and gel filtration, was 110 000 and 100 000, respectively. The pH optimum of this enzyme was approximately 9.0, while the Km for allophanate was 0.55 mM. Allophanate hydrolase was sensitive to N-ethylmaleimide but was protected from this inhibition by allophanate. Malonic acid, oxaloacetic acid, and acetoacetic acid were inhibitory to allophanate hydrolysis. 相似文献
17.
Boto AN Xu W Jakoncic J Pannuri A Romeo T Bessman MJ Gabelli SB Amzel LM 《Proteins》2011,79(8):2455-2466
The Nudix hydrolase superfamily, characterized by the presence of the signature sequence GX(5)EX(7)REUXEEXGU (where U is I, L, or V), is a well-studied family in which relations have been established between primary sequence and substrate specificity for many members. For example, enzymes that hydrolyze the diphosphate linkage of ADP-ribose are characterized by having a proline 15 amino acids C-terminal of the Nudix signature sequence. GDPMK is a Nudix enzyme that conserves this characteristic proline but uses GDP-mannose as the preferred substrate. By investigating the structure of the GDPMK alone, bound to magnesium, and bound to substrate, the structural basis for this divergent substrate specificity and a new rule was identified by which ADP-ribose pyrophosphatases can be distinguished from purine-DP-mannose pyrophosphatases from primary sequence alone. Kinetic and mutagenesis studies showed that GDPMK hydrolysis does not rely on a single glutamate as the catalytic base. Instead, catalysis is dependent on residues that coordinate the magnesium ions and residues that position the substrate properly for catalysis. GDPMK was thought to play a role in biofilm formation because of its upregulation in response to RcsC signaling; however, GDPMK knockout strains show no defect in their capacity of forming biofilms. 相似文献
18.
Glycoside hydrolase family 57 glycogen branching enzymes (GH57GBE) catalyze the formation of an α-1,6 glycosidic bond between α-1,4 linked glucooliogosaccharides. As an atypical family, a limited number of GH57GBEs have been biochemically characterized so far. This study aimed at acquiring a better understanding of the GH57GBE family by a systematic sequence-based bioinformatics analysis of almost 2500 gene sequences and determining the branching activity of several native and mutant GH57GBEs. A correlation was found in a very low or even no branching activity with the absence of a flexible loop, a tyrosine at the loop tip, and two β-strands. 相似文献
19.
Dimitra Triantafillidou Maria Simitsopoulou Francois Franceschi Theodora Choli-Papadopoulou 《Journal of Protein Chemistry》1999,18(2):215-223
The L11 ribosomal protein from Thermus thermophilus (TthL11) has been overproduced and purified to homogeneity using a two-step purification protocol. The overproduced protein carries a similar methylation pattern at Lys-3 as does its homolog from Escherichia coli. Chymotrypsin digested only a small part of the TthL11 protein and did not cleave TthL11 into two peptides, as in the case of EcoL11, but produced only a single N-terminal peptide. Tryptic digestion of TthL11 also produced an N-terminal peptide, in contrast to the C-terminal peptide obtained with L11 from Bacillus stearothermophilus. The recombinant protein forms a specific complex with a 55-nt 23S rRNA fragment known to interact with members of the L11 family from several organisms. Cooperative binding of TthL11 and thiostrepton to 23S rRNA leads to an increased protection of TthL11 from tryptic digestion. The similar structural and biochemical properties as well as the significant homology between L11 from E. coli and B. stearothermophilus with the corresponding protein from Thermus thermophilus indicate an evolutionarily conserved protein important for ribosome function. 相似文献