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1.
《Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology》1990,1037(2):133-139
The Met-168 residue in penicillin acylase from Kluyvera citrophila was changed to Ala by oligonucleotide site-directed mutagenesis. The Ala-168 mutant exhibited different substrate specificity than wild-type and enhanced thermal stability. The thermodynamic profiles for penicillin G hydrolysis catalyzed by both enzymes were obtained from the temperature dependence of the steady-state kinetic parameters Km and kcat. The high values of enthalpy and entropy of activation determined for the binding of substrate suggest that an induced-fit-like mechanism takes place. The Met→Ala168 mutation unstabilizes the first transition-state (E··S≠) and the enzyme-substrate complex (ES) causing a decrease in association equilibrium and specificity constants in the enzyme. However, no change is observed in the acyl-enzyme formation. It is concluded that residue 168 is involved in the enzyme conformational rearrangements caused by the interaction of the acid moiety of the substrate at the active site. 相似文献
2.
Substitution of the amino acid at position 102 with polar and aromatic residues influences substrate specificity of lactate dehydrogenase 总被引:1,自引:0,他引:1
David J. Nicholls Margaret Davey Susan E. Jones Julie Miller J. John Holbrook Anthony R. Clarke Michael D. Scawen Tony Atkinson Christopher R. Goward 《Journal of Protein Chemistry》1994,13(1):129-133
The Gin residue at amino acid position 102 ofBacillus stearothermophilus lactate dehydrogenase was replaced with Ser, Thr, Tyr, or Phe to investigate the effect on substrate recognition. The Q102S and Q102T mutant enzymes were found to have a broader range of substrate specificity (measured byk
cat/K
m) than the wild-type enzyme. However, it is evident that either Ser or Thr at position 102 are of a size able to accommodate a wide variety of substrates in the active site and substrate specificity appears to rely largely on size discrimination in these mutants. The Q102F and Q102Y mutant enzymes have low catalytic efficiency and do not show this relaxed substrate specificity. However, their activities are restored by the presence of an aromatic substrate. All of the enzymes have a very low catalytic efficiency with branched chain aliphatic substrates.Abbreviations used BSLDH
Bacillus stearothermophilus lactate dehydrogenase
- FBP
fructose-1,6-bisphosphate
- HP
hydroxypyruvate
- KB
ketobutyrate
- KC
ketocaproate
- KV
ketovalerate
- MDH
malate dehydrogenase
- PP
phenylpyruvate
- PYR
pyruvate
- RBE
relative binding energy 相似文献
3.
Engineering Streptomyces clavuligerus Deacetoxycephalosporin C Synthase for Optimal Ring Expansion Activity toward Penicillin G
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Chia-Li Wei Yunn-Bor Yang Wen-Ching Wang Wen-Chi Liu Jyh-Shing Hsu Ying-Chieh Tsai 《Applied microbiology》2003,69(4):2306-2312
The deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus was engineered with the aim of enhancing the conversion of penicillin G into phenylacetyl-7-aminodeacetoxycephalosporanic acid, a precursor of 7-aminodeacetoxycephalosporanic acid, for industrial application. A single round of random mutagenesis followed by the screening of 5,500 clones identified three mutants, G79E, V275I, and C281Y, that showed a two- to sixfold increase in the kcat/Km ratio compared to the wild-type enzyme. Site-directed mutagenesis to modify residues surrounding the substrate resulted in three mutants, N304K, I305L, and I305M, with 6- to 14-fold-increased kcat/Km values. When mutants containing all possible combinations of these six sites were generated to optimize the ring expansion activity for penicillin G, the double mutant, YS67 (V275I, I305M), showed a significant 32-fold increase in the kcat/Km ratio and a 5-fold increase in relative activity for penicillin G, while the triple mutant, YS81 (V275I, C281Y, I305M), showed an even greater 13-fold increase in relative activity toward penicillin G. Our results demonstrate that this is a robust approach to the modification of DAOCS for an optimized DAOCS-penicillin G reaction. 相似文献
4.
B. S. Deshpande S. S. Ambedkar J. G. Shewale 《World journal of microbiology & biotechnology》1996,12(4):373-378
Aeromonas sp. ACY 95 produces constitutively and intracellularly a penicillin V acylase at an early stage of fermentation (12 h) and a cephalosporin C acylase at a later stage (36 h). Some penicillins, cephalosporin C and their side chain moieties/analogues, phenoxyacetic acid, penicillin V and penicillin G, enhanced penicillin V acylase production while none of the test compounds affected cephalosporin C acylase production. Supplementation of the medium with some sugars and sugar derivatives repressed enzyme production to varying degrees. The studies on enzyme formation, induction and repression, and substrate profile suggest that the cephalosporin C acylase and penicillin V acylase are two distinct enzymes. Substrate specificity studies indicate that the Aeromonas sp. ACY 95 produces a true cephalosporin C acylase which unlike the enzymes reported hitherto hydrolyses cephalosporin C specifically.The authors are with Research and Development, Hindustan Antibiotics Limited, Pimpri. Pune 411 018, India 相似文献
5.
The smallest and enzymatically active molecule, TetApuQ818, was localized within the C-terminal Q818 amino acid residue after
serial C-terminal truncation analysis of the recombinant amylopullulanase molecule (TetApuM955) from Thermoanaerobacter pseudoethanolicus. Kinetic analyses indicated that the overall catalytic efficiency, k
cat/K
m, of TetApuQ818 was 8–32% decreased for the pullulan and the soluble starch substrate, respectively. Changes to the substrate
affinity, K
m, and the turnover rate, k
cat, were decreased significantly in both enzymatic activities of TetApuQ818. TetApuQ818 exhibited less thermostability than
TetApuM955 when the temperature was raised above 85°C, but it had similar substrate-binding ability and hydrolysis products
toward various substrates as TetApuM955 did. Both enzymes showed similar spectroscopies of fluorescence and circular dichroism,
suggesting the active folding conformation was maintained after this C-terminal Q818 deletion. This study suggested that the
binding ability of insoluble starch by TetApuM955 did not rely on the putative C-terminal carbohydrate binding module family
20 (CBM20) and two FnIII regions of TetApu, though the integrity of the AamyC module of TetApuQ818 was required for the enzyme
activity. 相似文献
6.
Fred A. Exterkate 《Applied microbiology and biotechnology》1990,33(4):401-406
Various chromophoric peptides have been tested as substrates for two genetically related types (PI and PIII) of cell-envelope proteinases of Lactococcus lactis subsp. cremoris. The positively charged peptide methoxy-succinyl-arginyl-prolyl-tyrosyl-p-nitroanilide appeared to be cleaved with the highest catalytic efficiency by both enzymes, although in the case of PIII only at high ionic strength. A cation binding site in the PI-type proteinase that is not present in the related PIII-type appears to be mainly responsible for the difference between these enzymes with respect to the rate of conversion of this chromophoric substrate at relatively low ionic strength. This cation binding site most probably resides in the aspartic acid residue 166,which in PIII is substituted by asparagine. Substitution of the threonine residue 138 by lysine in PIII may also play a role. The binding step in the reactionpathway catalysed by PI at low ionic strength is governed mainly by an ionic interaction involving the cation binding site. In addition, hydrophobic interactions contribute to the binding process. Masking of the cation binding site only increases the Michaelis constant K
m; the catalytic constant k
catis not affected. In the absence of the cation binding site (viz. in PIII) the free energy derived from the hydrophobic interactions only is too small to promote binding of the substrate effectively. High activities are measured only if a high ionic strength is introduced. Removal of electrostatic repulsion between the substrate and positively charged residues of the enzyme, among which is lysine 138, may contribute to this activation. Inhibition by n-butanol suggests the presence of an essential hydrophobic (binding) site which is primarily involved in the orientation of the substrate molecule for the catalytic reaction to be initiated. 相似文献
7.
Thermodynamic profiles of penicillin G hydrolysis catalyzed by wild-type and Met----Ala168 mutant penicillin acylases from Kluyvera citrophila 总被引:2,自引:0,他引:2
J Martín I Prieto J L Barbero J Pérez-Gil J M Manche?o R Arche 《Biochimica et biophysica acta》1990,1037(2):133-139
The Met-168 residue in penicillin acylase from Kluyvera citrophila was changed to Ala by oligonucleotide site-directed mutagenesis. The Ala-168 mutant exhibited different substrate specificity than wild-type and enhanced thermal stability. The thermodynamic profiles for penicillin G hydrolysis catalyzed by both enzymes were obtained from the temperature dependence of the steady-state kinetic parameters Km and kcat. The high values of enthalpy and entropy of activation determined for the binding of substrate suggest that an induced-fit-like mechanism takes place. The Met----Ala168 mutation unstabilizes the first transition-state (E..S not equal to) and the enzyme-substrate complex (ES) causing a decrease in association equilibrium and specificity constants in the enzyme. However, no change is observed in the acyl-enzyme formation. It is concluded that residue 168 is involved in the enzyme conformational rearrangements caused by the interaction of the acid moiety of the substrate at the active site. 相似文献
8.
A gluco‐oligosaccharide oxidase (GOOX) from Acremonium strictum type strain CBS 346.70 was cloned and expressed in Pichia pastoris. The recombinant protein, GOOX‐VN, contained fifteen amino acid substitutions compared with the previously reported A. strictum GOOX. These two enzymes share 97% sequence identity; however, only GOOX‐VN oxidized xylose, galactose, and N‐acetylglucosamine. Besides monosaccharides, GOOX‐VN oxidized xylo‐oligosaccharides, including xylobiose and xylotriose with similar catalytic efficiency as for cello‐oligosaccharides. Of three mutant enzymes that were created in GOOX‐VN to improve substrate specificity, Y300A and Y300N doubled kcat values for monosaccharide and oligosaccharide substrates. With this novel substrate specificity, GOOX‐VN and its variants are particularly valuable for oxidative modification of cello‐ and xylo‐oligosaccharides. Biotechnol. Bioeng. 2011;108: 2261–2269. © 2011 Wiley Periodicals, Inc. 相似文献
9.
Partially purified penicillin acylases (EC 3.5.1.11) were prepared from Pseudomonas melanogenum KY 3987 and Kluyvera citrophila KY 3641 capable of synthesizing d(–)-α-amino-benzylpenicillin (APc) from 6-aminopenicillanic acid (6-APA) and phenylglycine methyl ester. As the cell-free extract of P. melanogenum contained high levels of penicillinase (EC 3.5.2.6), the acylase was separated completely from the penicillinase by use of Sephadex column chromatography or electrofocusing. The most salient property of the P. melanogenum penicillin acylase was its substrate specificity to penicillin substrates: it could form 6-APA only from APc but not from penicillin G, penicillin V and p-aminobenzylpenicillin, whereas the K. citrophila acylase acted on all of these penicillins. The P. melanogenum enzyme is hence considered a novel type of penicillin acylase. 相似文献
10.
Human arylamine N-acetyltransferase 1 (NAT1) has been overexpressed in E. coli as a mutant dihydrofolic acid reductase (DHFR) fusion protein with a thrombin sensitive linker. An initial DEAE anion-exchange chromatography resulted in partial purification of the fusion protein. The fusion protein was cleaved with thrombin, and human rNAT1 was purified with a second DEAE column. A total of 8 mg of human rNAT1 from 2 l of cell culture was purified to homogeneity with this methodology. Arylamine substrate specificities were determined for human rNAT1 and hamster rNAT2. With both NATs, the second order rate constants (kcat/Kmb) for p-aminobenzoic acid (PABA) and 2-aminofluorene (2-AF) were several thousand-fold higher than those for procainamide (PA), consistent with the expected substrate specificities of the enzymes. However, p-aminosalicylic acid (PAS), previously reported to be a human NAT1 and hamster NAT2 selective substrate, exhibits 20-fold higher specificity for hamster rNAT2 (k cat/Kmb3410 M-1 s-1 ) than for human rNAT1 (kcat/Kmb 169.4 M-1 s-1 ). p-aminobenzoylglutamic acid (pABglu) was acetylated 10-fold more efficiently by human rNAT1 than by hamster rNAT2. Inhibition studies of human rNAT1 and hamster rNAT2 revealed that folic acid and methotrexate (MTX) are competitive inhibitors of both the unacetylated and acetylated forms of the enzymes, with KI values in 50–300 range. Dihydrofolic acid (DHF) was a much poorer inhibitor of human rNAT1 than of hamster rNAT2. The combined results demonstrate that human rNAT1 and hamster rNAT2 have similar but distinct kinetic properties with certain substrates, and suggest that folic acid, at least in the non-polyglutamate form, may not have an effect on human NAT1 activity in vivo. 相似文献
11.
Eliot T. Smith David A. Johnson 《Protein science : a publication of the Protein Society》2013,22(5):577-585
The serine protease enteropeptidase exhibits a high level of substrate specificity for the cleavage sequence DDDDK~ X, making this enzyme a useful tool for the separation of recombinant protein fusion domains. In an effort to improve the utility of enteropeptidase for processing fusion proteins and to better understand its structure and function, two substitution variants of human enteropeptidase, designated R96Q and Y174R, were created and produced as active (>92%) enzymes secreted by Pichia pastoris with yields in excess of 1.7 mg/Liter. The Y174R variant showed improved specificities for substrates containing the sequences DDDDK (kcat/KM = 6.83 × 106 M?1 sec?1) and DDDDR (kcat/KM = 1.89 × 107 M?1 sec?1) relative to all other enteropeptidase variants reported to date. BPTI inhibition of Y174R was significantly decreased. Kinetic data demonstrate the important contribution of the positively charged residue 96 to extended substrate specificity in human enteropeptidase. Modeling shows the importance of the charge–charge interactions in the extended substrate binding pocket. 相似文献
12.
Substrate specificity characterization for eight putative nudix hydrolases. Evaluation of criteria for substrate identification within the Nudix family
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Vi N. Nguyen Annsea Park Anting Xu John R. Srouji Steven E. Brenner Jack F. Kirsch 《Proteins》2016,84(12):1810-1822
The nearly 50,000 known Nudix proteins have a diverse array of functions, of which the most extensively studied is the catalyzed hydrolysis of aberrant nucleotide triphosphates. The functions of 171 Nudix proteins have been characterized to some degree, although physiological relevance of the assayed activities has not always been conclusively demonstrated. We investigated substrate specificity for eight structurally characterized Nudix proteins, whose functions were unknown. These proteins were screened for hydrolase activity against a 74‐compound library of known Nudix enzyme substrates. We found substrates for four enzymes with kcat/Km values >10,000 M?1 s?1: Q92EH0_LISIN of Listeria innocua serovar 6a against ADP‐ribose, Q5LBB1_BACFN of Bacillus fragilis against 5‐Me‐CTP, and Q0TTC5_CLOP1 and Q0TS82_CLOP1 of Clostridium perfringens against 8‐oxo‐dATP and 3'‐dGTP, respectively. To ascertain whether these identified substrates were physiologically relevant, we surveyed all reported Nudix hydrolytic activities against NTPs. Twenty‐two Nudix enzymes are reported to have activity against canonical NTPs. With a single exception, we find that the reported kcat/Km values exhibited against these canonical substrates are well under 105 M?1 s?1. By contrast, several Nudix enzymes show much larger kcat/Km values (in the range of 105 to >107 M?1 s?1) against noncanonical NTPs. We therefore conclude that hydrolytic activities exhibited by these enzymes against canonical NTPs are not likely their physiological function, but rather the result of unavoidable collateral damage occasioned by the enzymes' inability to distinguish completely between similar substrate structures. Proteins 2016; 84:1810–1822. © 2016 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc. 相似文献
13.
Hideyuki Suzuki Professor Chiaki Yamada Kyoko Kijima Sayaka Ishihara Kei Wada Keiichi Fukuyama Hidehiko Kumagai 《Biotechnology journal》2010,5(8):829-837
Semisynthetic cephalosporins, the best-selling antibiotics worldwide, are derived from 7-aminocephalosporanic acid (7-ACA). Currently, in the pharmaceutical industrie, 7-ACA is mainly produced from cephalosporin C by sequential application of D -amino acid oxidase and cephalosporin acylase. Here we study the potential of industrially amenable enzyme γ-glutamyltranspeptidase from Bacillus subtilis for 7-ACA production, since the wild-type γ-glutamyltranspeptidase of B. subtilis has inherent glutaryl-7-aminocephalosporanic acid acylase activity with a kcat value of 0.0485 s-1. Its activity has been enhanced by site directed and random mutagenesis. The kcat/Km value was increased to 3.41 s-1 mM-1 for a E423Y/E442Q/D445N mutant enzyme and the kcat value was increased to 0.508 s-1 for a D445G mutant enzyme. Consequently, the catalytic efficiency and the turnover rate were improved up to about 1000-fold and 10-fold, compared with the wildtype γ-glutamyltranspeptidase of B. subtilis. 相似文献
14.
High-performance liquid chromatography mass spectrometry (HPLC MS) was employed to assess the binding behaviors of various
substrates to Vibrio harveyi chitinase A. Quantitative analysis revealed that hexaNAG preferred subsites −2 to +2 over subsites −3 to +2 and pentaNAG
only required subsites −2 to +2, while subsites −4 to +2 were not used at all by both substrates. The results suggested that
binding of the chitooligosaccharides to the enzyme essentially occurred in compulsory fashion. The symmetrical binding mode
(−2 to +2) was favored presumably to allow the natural form of sugars to be utilized effectively. Crystalline α chitin was
initially hydrolyzed into a diverse ensemble of chitin oligomers, providing a clear sign of random attacks that took place
within chitin chains. However, the progressive degradation was shown to occur in greater extent at later time to complete
hydrolysis. The effect of the reducing-end residues were also investigated by means of HPLC MS. Substitutions of Trp275 to
Gly and Trp397 to Phe significantly shifted the anomer selectivity of the enzyme toward β substrates. The Trp275 mutation
modulated the kinetic property of the enzyme by decreasing the catalytic constant (k
cat) and the substrate specificity (k
cat/K
m) toward all substrates by five- to tenfold. In contrast, the Trp397 mutation weakened the binding strength at subsite (+2),
thereby speeding up the rate of the enzymatic cleavage toward soluble substrates but slowing down the rate of the progressive
degradation toward insoluble chitin. 相似文献
15.
Takayoshi Tagami Masayuki Okuyama Hiroyuki Nakai Young-Min Kim Haruhide Mori Kazunori Taguchi Birte Svensson Atsuo Kimura 《Biochimica et Biophysica Acta - Proteins and Proteomics》2013,1834(1):329-335
Glycoside hydrolase family 31 α-glucosidases (31AGs) show various specificities for maltooligosaccharides according to chain length. Aspergillus niger α-glucosidase (ANG) is specific for short-chain substrates with the highest kcat/Km for maltotriose, while sugar beet α-glucosidase (SBG) prefers long-chain substrates and soluble starch. Multiple sequence alignment of 31AGs indicated a high degree of diversity at the long loop (N-loop), which forms one wall of the active pocket. Mutations of Phe236 in the N-loop of SBG (F236A/S) decreased kcat/Km values for substrates longer than maltose. Providing a phenylalanine residue at a similar position in ANG (T228F) altered the kcat/Km values for maltooligosaccharides compared with wild-type ANG, i.e., the mutant enzyme showed the highest kcat/Km value for maltotetraose. Subsite affinity analysis indicated that modification of subsite affinities at + 2 and + 3 caused alterations of substrate specificity in the mutant enzymes. These results indicated that the aromatic residue in the N-loop contributes to determining the chain-length specificity of 31AGs. 相似文献
16.
Jin-Ha Kim Ponnandy Prabhu Marimuthu Jeya Manish Kumar Tiwari Hee-Jung Moon Raushan Kumar Singh Jung-Kul Lee 《Applied microbiology and biotechnology》2010,85(6):1839-1847
An isolated gene from Bacillus subtilis str. 168 encoding a putative isomerase was proposed as an L-arabinose isomerase (L-AI), cloned into Escherichia coli, and its nucleotide sequence was determined. DNA sequence analysis revealed an open reading frame of 1,491 bp, capable of
encoding a polypeptide of 496 amino acid residues. The gene was overexpressed in E. coli and the protein was purified using nickel-nitrilotriacetic acid chromatography. The purified enzyme showed the highest catalytic
efficiency ever reported, with a k
cat of 14,504 min−1 and a k
cat/K
m of 121 min−1 mM−1 for L-arabinose. A homology model of B. subtilis L-AI was constructed based on the X-ray crystal structure of E. coli
L-AI. Molecular dynamics simulation studies of the enzyme with the natural substrate, L-arabinose, and an analogue, D-galactose, shed light on the unique substrate specificity displayed by B. subtilis
L-AI only towards L-arabinose. Although L-AIs have been characterized from several other sources, B. subtilis
L-AI is distinguished from other L-AIs by its high substrate specificity and catalytic efficiency for L-arabinose. 相似文献
17.
Summary The stereoselectivity of penicillin amidase (PA, EC 3.5.1.11) from E coli and homologeous enzymes from other sources has been determined as a function of temperature and substrate for hydrolysis and kinetically controlled synthesis. The stereoselectivity of these reactions decreased almost by one order of magnitude from 5 to 45°C. It increased with the substrate (k
cat/K
m) and nucleophile (k
T/k
H) specificity, and was found to differ in the S1- (R-specific) and S1-(S-specific)-binding subsites of the active site. The S1-stereoselectivity was determined mainly by differences in the activation energy, i.e. the turnover number. The stereoselectivity of PA from different sources differed by almost an order of magnitude for the same substrate. 相似文献
18.
《Bioorganic & medicinal chemistry letters》2019,29(17):2452-2454
Protein tyrosine phosphatase (PTP) targeted, peptide based chemical probes are valuable tools for studying this important family of enzymes, despite the inherent difficulty of developing peptides targeted towards an individual PTP. Here, we have taken a rational approach to designing a SHP-2 targeted, fluorogenic peptide substrate based on information about the potential biological substrates of SHP-2. The fluorogenic, phosphotyrosine mimetic phosphocoumaryl aminopropionic acid (pCAP) provides a facile readout for monitoring PTP activity. By optimizing the amino acids surrounding the pCAP residue, we obtained a substrate with the sequence Ac-DDPI-pCAP-DVLD-NH2 and optimized kinetic parameters (kcat = 0.059 ± 0.008 s−1, Km = 220 ± 50 µM, kcat/Km of 270 M−1s−1). In comparison, the phosphorylated coumarin moiety alone is an exceedingly poor substrate for SHP-2, with a kcat value of 0.0038 ± 0.0003 s−1, a Km value of 1100 ± 100 µM and a kcat/Km of 3 M−1s−1. Furthermore, this optimized peptide has selectivity for SHP-2 over HePTP, MEG1 and PTPµ. The data presented here demonstrate that PTP-targeted peptide substrates can be obtained by optimizing the sequence of a pCAP containing peptide. 相似文献
19.
J. R. S. Alves L. P. Fonseca M. T. Ramalho J. M. S. Cabral 《Biotechnology Techniques》1995,9(10):729-730
Summary The synthetic substrate 6-nitro-3-(phenylacetamido) benzoic acid (NIPAB) is an appropriate substrate for assaying penicillin acylase activity in reversed micellar systems of Aerosol - OT in isooctane. Accumulation of 6-nitro-3-aminobenzoic acid (NABA) produced by the enzymatic hydrolysis of NIPAB, followed by the increase in absorbance at 405 nm, was linear at 4 to 20 mM for up to 30 minutes and 15 °C to 40 °C.Abbreviations PA
penicillin acylase (penicillin amidohydrolase EC 3.5.1.11)
- AOT
Aerosol OT (sodium bis- (2-ethylhexyl) sulfosuccinate)
- NIPAB
6-nitro-3-(phenylacetamido)-benzoic acid
- NABA
6-nitro-3-aminobenzoic acid
- BSA
bovine serum albumin 相似文献
20.
《DNA Repair》2017
The base excision repair DNA glycosylases, EcoNth and hNTHL1, are homologous, with reported overlapping yet different substrate specificities. The catalytic amino acid residues are known and are identical between the two enzymes although the exact structures of the substrate binding pockets remain to be determined. We sought to explore the sequence basis of substrate differences using a phylogeny-based design of site-directed mutations. Mutations were made for each enzyme in the vicinity of the active site and we examined these variants for glycosylase and lyase activity. Single turnover kinetics were done on a subgroup of these, comparing activity on two lesions, 5,6-dihydrouracil and 5,6-dihydrothymine, with different opposite bases. We report that wild type hNTHL1 and EcoNth are remarkably alike with respect to the specificity of the glycosylase reaction, and although hNTHL1 is a much slower enzyme than EcoNth, the tighter binding of hNTHL1 compensates, resulting in similar kcat/Kd values for both enzymes with each of the substrates tested. For the hNTHL1 variant Gln287Ala, the specificity for substrates positioned opposite G is lost, but not that of substrates positioned opposite A, suggesting a discrimination role for this residue. The EcoNth Thr121 residue influences enzyme binding to DNA, as binding is significantly reduced with the Thr121Ala variant. Finally, we present evidence that hNTHL1 Asp144, unlike the analogous EcoNth residue Asp44, may be involved in resolving the glycosylase transition state. 相似文献