The application of product inhibition studies to one substrate–one product enzymic reactions

A graphical procedure is presented for distinguishing between possible mechanisms of one substrate-one product enzymic reactions.     

The steady-state kinetics of isotope exchange for one substrate–one product enzymic reactions

Steady-state kinetic equations for isotope exchange are derived for a number of one substrate-one product enzymic mechanisms in which two molecules of substrate or product can be combined with an enzyme molecule at the one time (e.g. allosteric mechanisms). The usual assumption, that the radioactive material is distributed among the substrate and product components according to a first-order law, is not valid. One can recognize whether isotope-exchange kinetics of an enzyme reaction follows first-order behaviour by using various initial concentrations of the labelled substance added to a mixture.     

Inhibition of glutamate racemase by substrate–product analogues

d-Glutamate is an essential biosynthetic building block of the peptidoglycans that encapsulate the bacterial cell wall. Glutamate racemase catalyzes the reversible formation of d-glutamate from l-glutamate and, hence, the enzyme is a potential therapeutic target. We show that the novel cyclic substrate–product analogue (R,S)-1-hydroxy-1-oxo-4-amino-4-carboxyphosphorinane is a modest, partial noncompetitive inhibitor of glutamate racemase from Fusobacterium nucleatum (FnGR), a pathogen responsible, in part, for periodontal disease and colorectal cancer (K_i = 3.1 ± 0.6 mM, cf. K_m = 1.41 ± 0.06 mM). The cyclic substrate–product analogue (R,S)-4-amino-4-carboxy-1,1-dioxotetrahydro-thiopyran was a weak inhibitor, giving only ∼30% inhibition at a concentration of 40 mM. The related cyclic substrate–product analogue 1,1-dioxo-tetrahydrothiopyran-4-one was a cooperative mixed-type inhibitor of FnGR (K_i = 18.4 ± 1.2 mM), while linear analogues were only weak inhibitors of the enzyme. For glutamate racemase, mimicking the structure of both enantiomeric substrates (substrate–product analogues) serves as a useful design strategy for developing inhibitors. The new cyclic compounds developed in the present study may serve as potential lead compounds for further development.     

Vitamin D substrate–product relationship in idiopathic hypercalciuria

Absorptive hypercalciuria (AH) is associated with elevated levels of 1,25-dihydroxyvitamin D (1,25(OH)₂D). While no increase of 1,25(OH)₂D after oral administration of 25-hydroxyvitamin D (25OHD) at high doses has been claimed in normal subjects, a substrate–product relationship has been reported in normal children, young people after UV irradiation, older persons, postmenopausal women, primary hyperparathyroidism, renal failure, osteomalacia, and sarcoidosis. No data of this relationship in AH is available. To investigate 25OHD-1,25(OH)₂D substrate–product relationship in AH, 161 AH patients (mean age 60.9 ± 11.7 years) and 110 age- and sex-matched controls (mean age 61.5 ± 12.4 years) were studied. In 57 controls and 52 AH subjects 25OHD-1,25(OH)₂D relationship in basal conditions and after 2-week oral 25OHD (25 μg/day) administration were evaluated. In basal conditions 25OHD and 1,25(OH)₂D were correlated in both, controls and AH; 25OHD treatment was followed by an increase in serum 25OHD and 1,25(OH)₂D in both groups. However, delta responses of 25OHD and 1,25(OH)₂D to 25OHD were higher in AH suggesting an enhanced activity of 1α-hydroxylase. In conclusion, the higher response of 1,25(OH)₂D after oral 25OHD in AH patients suggests a differential capacity between both groups in handling the increases in 1,25(OH)₂D.     

The studies on substrate,product and inhibitor binding to a wild-type and neuronopathic form of human acid-β-glucosidase

Gaucher disease is a lysosomal storage disorder caused by deficiency of human acid β-glucosidase. Recent x-ray structural elucidation of the enzyme alone and in the presence of its inhibitor was done, which provided an excellent template for further studies on the binding of substrate, product and inhibitor. To draw correlations between the clinical manifestation of the disease driven by point mutations, L444P and L444R, and the placement and function of putative S-binding sites, the presented theoretical studies were undertaken, which comprised of molecular dynamics and molecular docking methods. The obtained results indicate the D443 and D445 residues as extremely important for physiological functionality of an enzyme. They also show, although indirectly, that binding of the substrate is influenced by an interplay of E235 and E334 residues, constituting putative substrate binding site, and the region flanked by D435 and D445 residues. Figure The binding of an arbitrarily chosen structure of glucosylceramide (A), conduritol-β-epoxide (B), glucose (C) to the active site D443/D445 (A1, B1, C1) and E320/E340 (A2, B2, C2) of the wild-type structure of human acid-β-glucosidase. A1, B1, C1 blue mask represents the residues D443-D445; red mask represents the residue D444; A2, B2, C2 blue mask represents loop1 (Ser³⁴⁵-Glu³⁴⁹) and loop2 (Val³⁹⁴-Asp³⁹⁹), whereas red mask the residues E235 and 340     

Rabbit liver contains one major sterol 12α-hydroxylase with broad substrate specificity

Conversion of cholesterol into cholic acid in mammalian liver requires a 12α-hydroxylation step. Results have been presented suggesting that two different enzymes are involved in this hydroxylation with different activities towards the two steroids believed to be the physiological substrates for the enzyme, 7α-hydroxy-4-cholesten-3-one and 5β-cholestane-3α,7α-diol. It is shown here that rabbit liver microsomes and partly purified sterol 12α-hydroxylase as well as COS cells transfected with a cDNA coding for this enzyme are able to catalyze 12α-hydroxylation of the two substrates at similar relative rates. Also 7α-hydroxycholesterol and 3α,7α-dihydroxy-5β-cholestanoic acid are 12α-hydroxylated by the three systems. It is concluded that rabbit liver contains one major sterol 12α-hydroxylase with a broad substrate specificity.     

Efficient production of (R)-(−)-mandelic acid with highly substrate/product tolerant and enantioselective nitrilase of recombinant Alcaligenes sp.

For efficient production of (R)-(−)-mandelic acid, a nitrilase gene from Alcaligenes sp. ECU0401 was cloned and overexpressed in Escherichia coli. After simple optimization of the culture conditions, the biocatalyst production was greatly increased from 500 to 7000 U/l. The recombinant E. coli whole cells showed strong tolerance against a high substrate concentration of up to 200 mM, and the concentration of (R)-(−)-mandelic acid after only 4 h of transformation reached 197 mM with an enantiomeric excess (ee_p) of 99%. In a fed-batch reaction with 600 mM mandelonitrile as the substrate, the cumulative production of (R)-(−)-mandelic acid after 17.5 h of conversion reached 520 mM. The recombinant E. coli cells could also be repeatedly used in the biotransformation, retaining 40% of the initial activity after 10 batches of reaction. The highly substrate/product tolerable and enantioselective nature of this recombinant nitrilase suggests that it is of great potential for the practical production of optically pure (R)-(−)-mandelic acid.     

Molecular determinants for substrate selectivity of ω-transaminases

ω-Transaminase (ω-TA) is an industrially important enzyme for production of chiral amines. About 20 (S)-specific ω-TAs known to date show remarkably similar substrate selectivity characterized by stringent steric constraint precluding entry of a substituent larger than an ethyl group in the small binding pocket (S) and dual recognition of an aromatic substituent as well as a carboxylate group in the large pocket (L). The strictly defined substrate selectivity of the available ω-TAs remains a limiting factor in the production of structurally diverse chiral amines. In this work, we cloned, purified, and characterized three new ω-TAs from Ochrobactrum anthropi, Acinetobacter baumannii, and Acetobacter pasteurianus that were identified by a BLASTP search using the previously studied ω-TA from Paracoccus denitrificans. All the new ω-TAs exhibited similar substrate specificity, which led us to explore whether the molecular determinants for the substrate specificity are conserved among the ω-TAs. To this end, key active site residues were identified by docking simulation using the X-ray structure of the ω-TA from Pseudomonas putida. We found that the dual recognition in the L pocket is ascribed to Tyr23, Phe88*, and Tyr152 for hydrophobic interaction and Arg414 for recognition of a carboxylate group. In addition, the docking simulation indicates that Trp60 and Ile262 form the S pocket where the substituent size up to an ethyl group turns out to be sterically allowed. The six key residues were found to be essentially conserved among nine ω-TA sequences, underlying the molecular basis for the high similarity in the substrate selectivity.     

Role of Glu445 in the substrate binding of β-glucosidase

A previously uncharacterized gene in Neosartorya fischeri was cloned and expressed in Escherichia coli. It was found to encode a β-glucosidase (NfBGL1) distinguishable from other BGLs by its high turnover of p-nitrophenyl β-d-glucopyranoside (pNPG). Molecular determinants for the substrate recognition of NfBGL1 were studied through an initial screening of residues by sequence alignment, a second screening by homology modeling and subsequent site-directed mutagenesis to alter individual screened residues. A conserved amino acid, E445, in the substrate binding pocket of wild-type NfBGL1 was identified as an important residue affecting substrate affinity. Replacement of E445 with amino acids other than aspartate significantly decreased the catalytic efficiency (k_cat/K_m) of NfBGL1 towards pNPG, mainly through decreased binding affinity. This was likely due to the disruption of hydrogen bonding between the substrate and the carboxylate oxygen of the residue at position 445. Density functional theory (DFT) based studies suggested that an acidic amino acid at position 445 raises the substrate affinity of NfBGL1 through hydrogen bonding. The residue E445 is completely conserved indicating that this position can be considered as a crucial determinant for the substrate binding among GHs tested.     

Conformational change in substrate binding, catalysis and product release: an open and shut case?

The role of conformational change in substrate binding, catalysis and product release is reviewed for 11 enzymes, for which crystal structures are available for the apo, substrate- and product-bound states. The extent of global conformational changes is measured, and the movements of the functional regions involved in catalysis and ligand binding are compared to the rest of the structure. We find that most of these enzymes undergo relatively small amounts of conformational change and particularly small changes in catalytic residue geometry, usually less than 1 A. In some enzymes there is significant movement of the binding residues, usually on surface loops.     

Donor substrate promiscuity of bacterial β1–3-N-acetylglucosaminyltransferases and acceptor substrate flexibility of β1–4-galactosyltransferases

β1–3-N-Acetylglucosaminyltransferases (β3GlcNAcTs) and β1–4-galactosyltransferases (β4GalTs) have been broadly used in enzymatic synthesis of N-acetyllactosamine (LacNAc)-containing oligosaccharides and glycoconjugates including poly-LacNAc, and lacto-N-neotetraose (LNnT) found in the milk of human and other mammals. In order to explore oligosaccharides and derivatives that can be synthesized by the combination of β3GlcNAcTs and β4GalTs, donor substrate specificity studies of two bacterial β3GlcNAcTs from Helicobacter pylori (Hpβ3GlcNAcT) and Neisseria meningitidis (NmLgtA), respectively, using a library of 39 sugar nucleotides were carried out. The two β3GlcNAcTs have complementary donor substrate promiscuity and 13 different trisaccharides were produced. They were used to investigate the acceptor substrate specificities of three β4GalTs from Neisseria meningitidis (NmLgtB), Helicobacter pylori (Hpβ4GalT), and bovine (Bβ4GalT), respectively. Ten of the 13 trisaccharides were shown to be tolerable acceptors for at least one of these β4GalTs. The application of NmLgtA in one-pot multienzyme (OPME) synthesis of two trisaccharides including GalNAcβ1–3Galβ1–4GlcβProN₃ and Galβ1–3Galβ1–4Glc was demonstrated. The study provides important information for using these glycosyltransferases as powerful catalysts in enzymatic and chemoenzymatic syntheses of oligosaccharides and derivatives which can be useful probes and reagents.     

Introducing a product sustainability budget at an automotive company—one option to increase the use of LCSA results in decision-making processes

The International Journal of Life Cycle Assessment - Product sustainability assessment should evaluate the impacts on all three dimensions of sustainability (environment, economy, and society)....     

How does a symmetric dimer recognize an asymmetric substrate? A substrate complex of HIV-1 protease

The crystal structure of an actual HIV-1 protease-substrate complex is presented at 2.0 A resolution (R-value of 19.7 % (R(free) 23.3 %)) between an inactive variant (D25N) of HIV-1 protease and a long substrate peptide, Lys-Ala-Arg-Val-Leu-Ala-Glu-Ala-Met-Ser, which covers a full binding epitope of capsid(CA)-p2, cleavage site. The substrate peptide is asymmetric in both size and charge distribution. To accommodate this asymmetry the two protease monomers adopt different conformations burying a total of 1038 A(2) of surface area at the protease-substrate interface. The specificity for the CA-p2 substrate peptide is mainly hydrophobic, as most of the hydrogen bonds are made with the backbone of the peptide substrate. Two water molecules bridge the two monomers through the loops Gly49-Gly52 (Gly49'-Gly52') and Pro79'-Val82' (Pro79-Val82). When other complexes are compared, the mobility of these loops is correlated with the content of the P1 and P1' sites. Interdependence of the conformational changes allows the protease to exhibit its wide range of substrate specificity.     

Enzyme–substrate complexes of allosteric citrate synthase: Evidence for a novel intermediate in substrate binding

The citrate synthase (CS) of Escherichia coli is an allosteric hexameric enzyme specifically inhibited by NADH. The crystal structure of wild type (WT) E. coli CS, determined by us previously, has no substrates bound, and part of the active site is in a highly mobile region that is shifted from the position needed for catalysis. The CS of Acetobacter aceti has a similar structure, but has been successfully crystallized with bound substrates: both oxaloacetic acid (OAA) and an analog of acetyl coenzyme A (AcCoA). We engineered a variant of E. coli CS wherein five amino acids in the mobile region have been replaced by those in the A. aceti sequence. The purified enzyme shows unusual kinetics with a low affinity for both substrates. Although the crystal structure without ligands is very similar to that of the WT enzyme (except in the mutated region), complexes are formed with both substrates and the allosteric inhibitor NADH. The complex with OAA in the active site identifies a novel OAA-binding residue, Arg306, which has no functional counterpart in other known CS-OAA complexes. This structure may represent an intermediate in a multi-step substrate binding process where Arg306 changes roles from OAA binding to AcCoA binding. The second complex has the substrate analog, S-carboxymethyl-coenzyme A, in the allosteric NADH-binding site and the AcCoA site is not formed. Additional CS variants unable to bind adenylates at the allosteric site show that this second complex is not a factor in positive allosteric activation of AcCoA binding.     

Identification of β-catenin as a novel substrate of polo-like kinase 1

Polo-like kinase 1 (Plk1) is a serine/threonine kinase that plays an important role in M phase progression by regulating various downstream substrates via phosphorylation. Here, we identified β-catenin as a novel substrate of Plk1 and determined that Ser-718 is a phosphorylation site for Plk1 by using a phospho-specific antibody that cross-reacts with Plk1-dependent phosphorylation sites. Ser-718 of β-catenin was directly phosphorylated by recombinant Plk1 in vitro, with the phosphorylation signal in cells increasing with overexpression of Plk1 and decreasing when endogenous Plk1 was depleted by small interfering RNA. The phosphorylation at Ser-718 was correlated with the cell cycle-dependent expression of Plk1 which reached a maximum in M phase. We also confirmed that there is a physical interaction between β-catenin and Plk1 using coimmunoprecipitation and a GST pull-down assay. These results demonstrate that β-catenin is a physiological substrate of Plk1 in cells, which may provide a novel insight into the role of β-catenin in M phase.     

Elimination of substrate inhibition of a β-N-acetyl-d-hexosaminidase by single site mutation

Substrate inhibition hinders chitinolytic β-N-acetyl-d-hexosaminidases in producing N-acetyl-d-glucosamine (GlcNAc), the valuable chemical widely applied in medical and food industries. Here we focused on a promising chitinolytic enzyme, OfHex1 from the insect, Ostrinia furnacalis. By structural analysis of OfHex1, five residues nearby the active pocket including V327, E328, Y471, V484 and W490 were chosen and nine mutants including V327G, E328Q, E328A, Y471V, V484R, W490A, W490H, V327G/V484R/W490A and V327G/Y471V/W490H were constructed and recombinantly expressed in Pichia pastoris. The best-performing mutant, W490A, obtained by a higher yield of 5 mg/L, did not show substrate inhibition even when 5 mM of the substrates, (GlcNAc)_2–4, were applied. The k_cat/K_m values for (GlcNAc)_2–4 are 239.8, 111.3 and 79.8 s^?1 mM^?1, respectively. Besides, the pH stability of the mutant ranges from pH 4 to 11 and the thermal stability is up to 50 °C. This work suggests the W490A mutant might be an ideal biocatalyst for GlcNAc production from chitin.