Phenylalanine 93 of the human UGT1A10 plays a major role in the interactions of the enzyme with estrogens

Little is currently known about the substrate binding site of the human UDP-glucuronosyltransferases (UGTs) and the structural elements that affect their complex substrate selectivity. In order to further understand and extend our earlier findings with phenylalanines 90 and 93 of UGT1A10, we have replaced each of them with Gly, Ala, Val, Leu, Ile or Tyr, and tested the activity of the resulting 12 mutants toward eight different substrates. Apart from scopoletin glucuronidation, the F90 mutants other than F90L were nearly inactive, while the F93 mutants’ activity was strongly substrate dependent. Hence, F93L displayed high entacapone and 1-naphthol glucuronidation rates, whereas F93G, which was nearly inactive in entacapone glucuronidation, was highly active toward estradiol, estriol and even ethinylestradiol, a synthetic estrogen that is a poor substrate for the wild-type UGT1A10. Kinetic analyses of 4-nitrophenol, estradiol and ethinylestradiol glucuronidation by the mutants that catalyzed the respective reactions at considerable rates, revealed increased K_m values for 4-nitrophenol and estradiol in all the mutants, whilst the K_m values of F93G and F93A for ethinylestradiol were lower than in control UGT1A10. Based on the activity results and a new molecular model of UGT1A10, it is suggested that both F90 and F93 are located in a surface helix at the far end of the substrate binding site. Nevertheless, only F93 directly affects the selectivity of UGT1A10 toward large and rigid estrogens, particularly those with substitutions at the D ring. The effects of F93 mutations on the glucuronidation of smaller or less rigid substrates are indirect, however.     

Cloning,Isolation, and Properties of a New Homologous Exoarabinase from the <Emphasis Type="Italic">Penicillium canescens</Emphasis> Fungus

A novel exo-arabinase (GH93, exo-ABN) enzyme produced by the ascomycete Penicillium canescens has been studied. Cloning of the abn1 gene coding for exo-ABN into the recipient P. canescens strain RN3-11-7 yielded recombinant producing strains characterized by a high yield of extracellular exo- ABN production (20–30% of the total amount of extracellular protein). Chromatographic purification yielded a homogenous exo-ABN with a molecular weight of 47 kDa, as shown by SDS-PAGE. The enzyme showed high specific activity towards linear arabinan (117 U/mg) and low specific activity towards branched arabinan and arabinoxylan (4–5 U/mg) and para-nitrophenyl-α-L-arabinofuranoside (0.3 U/mg), whereas arabinogalactan and para-nitrophenyl-α-L-arabinopyranoside, the substrates that contained the pyranose form of arabinose, were not hydrolyzed. Arabinohexaose was the major product of linear arabinan hydrolysis. Exo-ABN had a pH optimum at 5.0 and a temperature optimum at 60°C. The enzyme was stable in a broad pH range (4.0–7.0) and upon heating to 50°C during 180 min. Extensive hydrolysis of linear and branched arabinans by exo- and endo-arabinase mixtures, arabinofuranosidase, and arabinofuran-arabinoxylan hydrolase has been performed. The degree of substrate conversion amounted to 67 and 83% of the maximal possible value, respectively.     

Short N-terminal region of UDP-galactose transporter (SLC35A2) is crucial for galactosylation of N-glycans

UDP-galactose transporter (UGT) and UDP-N-acetylglucosamine transporter (NGT) form heterologous complexes in the Golgi apparatus (GA) membrane. We aimed to identify UGT region responsible for galactosylation of N-glycans. Chimeric proteins composed of human UGT and either NGT or CMP-sialic acid transporter (CST) localized to the GA, and all but UGT/CST chimera corrected galactosylation defect in UGT-deficient cell lines, although at different efficiency. Importantly, short N-terminal region composed of 35 N-terminal amino-acid residues of UGT was crucial for galactosylation of N-glycans. The remaining molecule must be derived from NGT not CST, confirming that the role played by UGT and NGT is coupled.     

Engineering Monolignol 4-O-Methyltransferases to Modulate Lignin Biosynthesis

Lignin is a complex polymer derived from the oxidative coupling of three classical monolignols. Lignin precursors are methylated exclusively at the meta-positions (i.e. 3/5-OH) of their phenyl rings by native O-methyltransferases, and are precluded from substitution of the para-hydroxyl (4-OH) position. Ostensibly, the para-hydroxyls of phenolics are critically important for oxidative coupling of phenoxy radicals to form polymers. Therefore, creating a 4-O-methyltransferase to substitute the para-hydroxyl of monolignols might well interfere with the synthesis of lignin. The phylogeny of plant phenolic O-methyltransferases points to the existence of a batch of evolutionarily “plastic” amino acid residues. Following one amino acid at a time path of directed evolution, and using the strategy of structure-based iterative site-saturation mutagenesis, we created a novel monolignol 4-O-methyltransferase from the enzyme responsible for methylating phenylpropenes. We show that two plastic residues in the active site of the parental enzyme are vital in dominating substrate discrimination. Mutations at either one of these separate the evolutionarily tightly linked properties of substrate specificity and regioselective methylation of native O-methyltransferase, thereby conferring the ability for para-methylation of the lignin monomeric precursors, primarily monolignols. Beneficial mutations at both sites have an additive effect. By further optimizing enzyme activity, we generated a triple mutant variant that may structurally constitute a novel phenolic substrate binding pocket, leading to its high binding affinity and catalytic efficiency on monolignols. The 4-O-methoxylation of monolignol efficiently impairs oxidative radical coupling in vitro, highlighting the potential for applying this novel enzyme in managing lignin polymerization in planta.     

Characterization of the acid phosphatase and arylsulphatase activities in a tumorous blood cell line of Drosophila melanogaster

The lysosomal acid phosphatase (AP) and arylsulphatase (AS) activities in a tumorous blood cell line of Drosophila melanogaster have been characterized. The AP activity measured with para-nitrophenylphosphate as substrate had a pH optimum of pH 4. The K_m for this substrate was 0.1 mM. The activity was inhibited by l-tartrate, fluoride ions, DFP and anions such as phosphate and molybdate, but not by sulphydryl reagents nor by alkaline phosphatase inhibitors. The activity was adsorbed onto DEAE-Sephadex at pH 8 and eluted as one peak, when a NaCl gradient was applied to the column. It was not possible to separate the activity into several components by disc-gel electrophoresis or isoelectric focusing (pI of AP: 6.2). The mobility during disc-gel electrophoresis was not altered by prior incubation with bacterial neuraminidase. The AS was shown to hydrolyse the artificial substrates para-nitrophenylsulphate, para-nitrocatecholsulphate and acetylphenyl sulphate with pH optima of 6.8–7.2. The activity was detrimentally affected by various inorganic anions and could be totally inhibited by low concentrations of AgNO₃ (10⁻⁴ M). The AS bound to the anion exchanger more strongly than the AP, but in common with the AP, only one peak of activity could be detected by this and other separation techniques. The pI of the AS was 4.6. The AP and AS have similar apparent molecular weights (60–65,000) and sedimentation coefficients (6.6 and 5.8S, respectively).     

Amino acid positions 69-132 of UGT1A9 are involved in the C-glucuronidation of phenylbutazone

Phenylbutazone (PB) is known to be biotransformed to its O- and C-glucuronide. Recently, we reported that PB C-glucuronide formation is catalyzed by UGT1A9. Interestingly, despite UGT1A8 sharing high amino acid sequence identity with UGT1A9, UGT1A8 had no PB C-glucuronidating activity. In the present study, we constructed eight UGT1A9/UGT1A8 chimeras and evaluated which region is important for PB C-glucuronide formation. All of the chimeras and UGT1A8 and UGT1A9 had 7-hydroxy-(4-trifluoromethyl)coumarin (HFC) O-glucuronidating activity. The K_m values for HFC glucuronidation of UGT1A8, UGT1A9 and their chimeras were divided into two types, UGT1A8 type (high K_m) and UGT1A9 type (low K_m), and these types were determined according to whether their amino acids at positions 69-132 were those of UGT1A8 or UGT1A9. Likewise, PB O-glucuronidating activity was also detected by all of the chimeras, and their K_m values were divided into two types. On the contrary, PB C-glucuronidating activity was detected by UGT1A9_(1-132)/1A8_(133-286), UGT1A9_(1-212)/1A8_(213-286), UGT1A8_(1-68)/1A9_(69-286), and UGT1A8_(1-68)/1A9_(69-132)/1A8_(133-286) chimeras. The region 1A9_(69-132) was common among chimeras having PB C-glucuronidating activity. Of interest is that UGT1A9_(1-68)/1A8_(69-132)/1A9_(133-286) had lost PB C-glucuronidation activity, but retained activities of PB and HFC O-glucuronidation. These results strongly suggested that amino acid positions 69-132 of UGT1A9 are responsible for chemoselectivity for PB and affinity to substrates such as PB and HFC.     

A para-nitrophenol phosphonate probe labels distinct serine hydrolases of Arabidopsis

Activity-based protein profiling represents a powerful methodology to probe the activity state of enzymes under various physiological conditions. Here we present the development of a para-nitrophenol phosphonate activity-based probe with structural similarities to the potent agrochemical paraoxon. We demonstrate that this probes labels distinct serine hydrolases with the carboxylesterase CXE12 as the predominant target in Arabidopsis thaliana. The designed probe features a distinct labeling pattern and therefore represents a promising chemical tool to investigate physiological roles of selected serine hydrolases such as CXE12 in plant biology.     

Enhanced activity of <Emphasis Type="Italic">Withania somnifera</Emphasis> family-1 glycosyltransferase (UGT73A16) via mutagenesis

This work used an approach of enzyme engineering towards the improved production of baicalin as well as alteration of acceptor and donor substrate preferences in UGT73A16. The 3D model of Withania somnifera family-1 glycosyltransferase (UGT73A16) was constructed based on the known crystal structures of plant UGTs. Structural and functional properties of UGT73A16 were investigated using docking and mutagenesis. The docking studies were performed to understand the key residues involved in substrate recognition. In the molecular model of UGT73A16, substrates binding pockets are located between N- and C-terminal domains. Modeled UGT73A16 was docked with UDP-glucose, UDP-glucuronic acid (UDPGA), kaempferol, isorhamnetin, 3-hydroxy flavones, naringenin, genistein and baicalein. The protein–ligand interactions showed that His 16, Asp 246, Lys 255, Ala 337, Gln 339, Val 340, Asn 358 and Glu 362 amino acid residues may be important for catalytic activity. The kinetic parameters indicated that mutants A337C and Q339A exhibited 2–3 fold and 6–7 fold more catalytic efficiency, respectively than wild type, and shifted the sugar donor specificity from UDP-glucose to UDPGA. The mutant Q379H displayed large loss of activity with UDP-glucose and UDPGA strongly suggested that last amino acid residue of PSPG box is important for glucuronosylation and glucosylation and highly specific to sugar binding sites. The information obtained from docking and mutational studies could be beneficial in future to engineer this biocatalyst for development of better ones.     

Complexes of disubstituted benzene positional isomers with α-cyclodextrin

Stability constants for complex formation between α-cyclodextrin and some ortho-, meta-, and para-disubstituted benzenes were measured in aqueous solution at 25°C by potentiometry, spectrophotometry, competitive spectrophotometry, and solubility. All systems form 1:1 complexes, some para substrates form 1:2 complexes (one substrate to two cyclodextrins), but no meta substrates form 1:2 complexes. Ortho substrates form weak complexes. These observations are accounted for in terms of a binding site molecular model. On the average over many systems, K₁₁ (para) and K₁₁ (meta) are approximately equal. Major discrepancies (greater than a factor of two) are diagnostic of significantly different electronic or steric effects in the complexing abilities of the isomeric substrates.     

A thrombin inhibitor from the gut of <Emphasis Type="Italic">Boophilus microplus</Emphasis> ticks

A thrombin inhibitor was identified for the first time in the gut of the cattle tick Boophilus microplus. Here we present the partial purification and characterization of this new molecule, which was purified from the gut extract by three chromatographic steps: ion-exchange, gel filtration and affinity chromatography in a thrombin–Sepharose resin. In SDS-PAGE the inhibitor showed an apparent molecular mass of circa 26 kDa, which is different from the two thrombin inhibitors present in the saliva of this tick. The new inhibitor delays bovine plasma clotting time and inhibits both thrombin induced fibrinogen clotting and thrombin induced platelet aggregation. However, it does not interfere with thrombin amidolytic activity upon a small substrate (H-D-Phe-Pip-Arg-para-nitroanilide), which does not require binding to thrombin exosites. Therefore, the inhibitor does not block thrombin active site, although it must interfere with one of the thrombin exosites. B. microplus gut thrombin inhibitor (BmGTI) is also capable of enhancing activated protein C (APC) activity upon its specific substrate (H-D-Glu-Pro-Arg-para-nitroanilide), an activity never described before among B. microplus molecules.     

Glucuronidation of haloperidol by rat liver microsomes: involvement of family 2 UDP-glucuronosyltransferases

The purposes of this study were to develop a HPLC method to assay for haloperidol glucuronide (HALG); to apply this assay method to the in vitro determination of haloperidol (HAL) UDP-glucuronosyltransferase (UGT) enzyme kinetics in rat liver microsomes (RLM); and to identify the UGT isoforms catalyzing glucuronidation of HAL in rats. Incubation of Brij-activated RLM with HAL and UDP-glucuronic acid (UDPGA) in TRIS pH 7.4 buffer resulted in the formation of a single peak in the HPLC chromatogram at 270 nm. The identity of this peak was confirmed to be that of HALG by 1) β-glucuronidase hydrolysis; 2) incubation without UDPGA; 3) UV spectral analysis; and 4) LC/MS/MS to yield the expected mass of 552.1. Enzyme kinetic studies using single enzyme Michaelis-Menton model showed an apparent V_max = 271.9 ± 10.1 pmoles min⁻¹ mg protein⁻¹ and K_m = 61 ± 7.2 μM. Glucuronidation activity in homozygous Gunn (j/j) rats was approximately 80% as compared to Sprague-Dawley RLM. HALG formation was approximately doubled in PB-induced RLM. There was no increase in glucuronidation activities in 3MC-induced RLM. The Gunn rat and the PB-induced RLM data suggest predominant but not exclusive involvement of the UGT2B family in the formation of HALG. Because the UGTs exhibit overlapping substrate specificities and most substrates are glucuronidated by more than one isoform, inhibition studies with UGT2B1 substrate probe testosterone and the UGT2B12 substrate probe borneol were conducted. UGT2B1 and UGT2B12 exhibited 40% and 90% inhibition of HAL glucuronidation, respectively. Thus, UGT2B12 and UGT 2B1 isoforms are responsible for catalyzing HAL glucuronidation in rats. Our HPLC assay provides a specific and sensitive technique for the measurement of in vitro HAL-UGT activity.     

Isolation and biochemical characteristics of a molecular form of epididymal acid phosphatase of boar seminal plasma

The fluid of boar epididymis is characterized by a high activity of acid phosphatase (AcP), which occurs in three molecular forms. An efficient procedure was developed for the purification of a molecular form of epididymal acid phosphatase from boar seminal plasma. We focused on the epididymal molecular form, which displayed the highest electrophoretic mobility. The purification procedure (dialysis, ion exchange chromatography, affinity chromatography and hydroxyapatite chromatography) used in this study gave more than 7000-fold purification of the enzyme with a yield of 50%. The purified enzyme was homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified molecular form of the enzyme is a thermostable 50 kDa glycoprotein, with a pI value of 7.1 and was highly resistant to inhibitors of acid phosphatase when p-nitrophenyl phosphate was used as the substrate. Hydrolysis of p-nitrophenyl phosphate by the purified enzyme was maximally active at pH of 4.3; however, high catalytic activity of the enzyme was within the pH range of 3.5–7.0. Kinetic analysis revealed that the purified enzyme exhibited affinity for phosphotyrosine (K_m = 2.1 × 10⁻³ M) and was inhibited, to some extent, by sodium orthovanadate, a phosphotyrosine phosphatase inhibitor. The N-terminal amino acid sequence of boar epididymal acid phosphatase is ELRFVTLVFR, which showed 90% homology with the sequence of human, mouse or rat prostatic acid phosphatase.The purification procedure described allows the identification of the specific biochemical properties of a molecular form of epididymal acid phosphatase, which plays an important role in the boar epididymis.     

Functional characterization of a UDP-glucose:flavonoid 3-O-glucosyltransferase from the seed coat of black soybean (Glycine max (L.) Merr.)

The seed coats of black soybean (Glycine max (L.) Merr.) accumulate red (cyanidin-), blue (delphinidin-), purple (petunidin-), and orange (pelargonidin-based) anthocyanins almost exclusively as 3-O-glucosides; however, the responsible enzyme has not been identified. In this study, the full-length cDNA which encodes the enzyme that catalyzes the final step in anthocyanin biosynthesis, namely UDP-glucose:flavonoid 3-O-glucosyltransferase (UGT78K1), was isolated from the seed coat tissue of black soybean using rapid amplification of cDNA ends (RACE). Of the 28 flavonoid substrates tested, the purified recombinant protein glucosylated only anthocyanidins and flavonols, and demonstrated strict 3-OH regiospecificity. Galactose could also be transferred with relatively low activity to the 3-position of cyanidin or delphinidin in vitro. These findings are consistent with previous reports of mainly 3-O-glucosylated and minor amounts of 3-O-galactosylated anthocyanins in the seed coat of black soybean. The recombinant enzyme exhibited pronounced substrate inhibition by cyanidin at 100 μM acceptor concentration. Transfer of UGT78K1 into the Arabidopsis T-DNA mutant (ugt78d2) deficient in anthocyanidin and flavonol 3-O-glucosyltransferase activity, restored the accumulation of anthocyanins and flavonols, suggesting the in vivo function of the enzyme as a flavonoid 3-O-glucosyltransferase. Genomic and phylogenetic analyses suggest the existence of three additional soybean sequences with high similarity to UGT78K1. RT-PCR confirmed the co-expression of one of these genes (Glyma08g07130) with UGT78K1 in the seed coat of black soybean, suggesting possible functional redundancies in anthocyanin biosynthesis in this tissue.     

Isolation and characterization of a novel Rhodococcus strain with switchable carbonyl reductase and para-acetylphenol hydroxylase activities

In the search for an effective biocatalyst for the reduction of acetophenones with unprotected hydroxy group on the benzene ring, a microorganism, which reduced para-acetylphenol to S-(?)-1-(para-hydroxyphenyl)ethanol under anaerobic conditions, was isolated from soil samples and the 16S rDNA study showed that it was phylogenetically affiliated with species of the genus Rhodococcus and was most similar to Rhodococcus pyridinivorans. Unexpectedly, this strain also hydroxylated para-acetylphenol to give 4-acetylcatechol in presence of oxygen, possessing para-acetylphenol hydroxylase activity. While the reduction of para-acetylphenol had an optimal reaction pH at 7 and a broad optimal temperature range (35–45 °C), the hydroxylation reached the maximum conversion at the pH range of 7–8 and 35 °C. This study identified for the first time a Rhodococcus strain with para-acetylphenol hydroxylase activity, which also contains highly enantioselective carbonyl reductase activity with potential applications for the asymmetric reduction of these less-explored but important ketones such as α-aminoacetophenone, 3′-hydroxyacetophenone and 4′-hydroxyacetophenone. The para-acetylphenol hydroxylase and carbonyl reductase activity are switchable by the reaction conditions.     

Genomic organization of a UDP-glucosyltransferase gene determines differential accumulation of specific flavonoid glucosides in tepals

Glycosylation plays a major role in the chemical diversity of flavonoids. The wide diversity of the family-1 glycosyltransferase (UGT) impairs the determination of the biochemical function solely from its primary sequence. Here we combined differential expression and target metabolomic analysis in various Crocus species to identify a gene that is key in determining the flavonoid composition of Crocus species that belong to the Crocus series. UGT703B1 recognizes isorhamnetin and kaempferol as substrates in vitro. In addition, UGT703B1 expression was found to be highly correlated with the presence of kaempferol 7-O-biglucoside-3-O-β-glucoside and isorhamnetin-3,7-O-diglucoside. These flavonols were present in C. sativus and C. cartwrightianus albus, both from series Crocus but absent in Crocus species from the other series analyzed. Further, the presence of both flavonols was associated with the expression of UGT703B1, and this expression was correlated with the presence of the UGT703B1 coding gene, with the exception of C. cancellatus, whose genomic sequence was present but contained a shorter intronic sequence and promoter alterations, suggesting the presence of regulatory sequences in the deleted part of that intron and promoter important for UGT703B1 expression. Overall, the data obtained supports the involvement of UGT703B1 in the formation of specific kaempferol and isorhamnetin glucosides, while demonstrating that the integration of metabolomic and differential expression analysis is a versatile tool for understanding a multigene family of UGTs in Crocus.     

Purification of S-2-hydroxyacylglutathione hydrolase (glyoxalase II) from rat erythrocytes.

Glyoxalase II, a specific glutathione thiolesterase, has been purified 9100-fold from rat erythrocytes using a purification scheme which employs Affi-Gel blue as a hydrophobic affinity column and also employs a glutathione-affinity column prepared by coupling S-(p-chlorophenacyl)glutathione to Affi-Gel 202. This procedure offers a convenient method for the preparation of highly purified glyoxalase II. Also described is a convenient method for the preparation of S-lactoyl-glutathione, a substrate for glyoxalase II.     

d-Aspartate N-methyltransferase catalyzes biosynthesis of N-methyl-d-aspartate (NMDA), a well-known selective agonist of the NMDA receptor,in mice

N-Methyl-d-aspartate (NMDA), which is a selective agonist for the NMDA receptor, has recently been shown to be present in various biological tissues. In mammals, the activity of d-aspartate N-methyltransferase (DDNMT), which produces NMDA from d-aspartate, has been detected only in homogenates prepared from rat tissues. Moreover, the enzymatic properties of DDNMT have been poorly studied and its molecular entity has not yet been identified. In this report, we show for the first time that the activity of DDNMT is present in mouse tissues and succeed in obtaining a partially purified enzyme preparation from a mouse tissue homogenate with a purification fold of 1900 or more, and have characterized the enzymatic activity of this preparation. The results indicate that DDNMT, which is highly specific for d-aspartate and is S-adenosyl-l-methionine-dependent, is a novel enzyme that clearly differs from the known methylamine-glutamate N-methyltransferase (EC 2.1.1.21) and glycine N-methyltransferase (EC 2.1.1.20).     

UGT86C11 is a novel plant UDP-glycosyltransferase involved in labdane diterpene biosynthesis

Glycosyltransferases constitute a large family of enzymes across all domains of life, but knowledge of their biochemical function remains largely incomplete, particularly in the context of plant specialized metabolism. The labdane diterpenes represent a large class of phytochemicals with many pharmacological benefits, such as anti-inflammatory, hepatoprotective, and anticarcinogenic. The medicinal plant kalmegh (Andrographis paniculata) produces bioactive labdane diterpenes; notably, the C19-hydroxyl diterpene (andrograpanin) is predominantly found as C19-O-glucoside (neoandrographolide), whereas diterpenes having additional hydroxylation(s) at C3 (14-deoxy-11,12-didehydroandrographolide) or C3 and C14 (andrographolide) are primarily detected as aglycones, signifying scaffold-selective C19-O-glucosylation of diterpenes in planta. Here, we analyzed UDP-glycosyltransferase (UGT) activity and diterpene levels across various developmental stages and tissues and found an apparent correlation of UGT activity with the spatiotemporal accumulation of neoandrographolide, the major diterpene C19-O-glucoside. The biochemical analysis of recombinant UGTs preferentially expressed in neoandrographolide-accumulating tissues identified a previously uncharacterized UGT86 member (ApUGT12/UGT86C11) that catalyzes C19-O-glucosylation of diterpenes with strict scaffold selectivity. ApUGT12 localized to the cytoplasm and catalyzed diterpene C19-O-glucosylation in planta. The substrate selectivity demonstrated by the recombinant ApUGT12 expressed in plant and bacterium hosts was comparable to native UGT activity. Recombinant ApUGT12 showed significantly higher catalytic efficiency using andrograpanin compared with 14-deoxy-11,12-didehydroandrographolide and trivial activity using andrographolide. Moreover, ApUGT12 silencing in plants led to a drastic reduction in neoandrographolide content and increased levels of andrograpanin. These data suggest the involvement of ApUGT12 in scaffold-selective C19-O-glucosylation of labdane diterpenes in plants. This knowledge of UGT86 function might help in developing plant chemotypes and synthesis of pharmacologically relevant diterpenes.     

Chromogenic substrate from 4-nitro-1-naphthol for hydrolytic enzyme of neutral or slightly acidic optimum pH: 4-Nitro-1-naphthyl-β-d-galactopyranoside as an example

At pH from 5.5 to 7.6, absorptivity of 4-nitro-1-naphthol at 450 nm is over 2.1-fold of that of para-nitrophenol at 405 nm and over 9.6-fold of that of ortho-nitrophenol at 415 nm. On 4-nitro-1-naphthyl-β-d-galactopyranoside at pH 7.4, catalytic efficiency of Escherichia coli β-d-galactosidase is 3-fold of that on para-nitrophenyl-β-d-galactopyranoside and about 40% of that on ortho-nitrophenyl-β-d-galactopyranoside, and produces a lower quantification limit of penicillin G by enzyme-linked-immunoabsorbent-assay. Hence, 4-nitro-1-naphthol is favorable to prepare chromogenic substrates of hydrolytic enzymes of neutral or slightly acidic optimum pH.