Mechanism Of Substrate Inhibition Research Articles

New insights into the substrate inhibition of human 17β-hydroxysteroid dehydrogenase type 1

Human type 1 17β-hydroxysteroid dehydrogenase (17β-HSD1),a member of the short-chain dehydrogenase/reductase family, catalyzes the last step in the bioactivation of the most potent estrogen estradiol with high specificity and is thus involved in estrogen-dependent diseases. As an oxidoreductase, 17β-HSD1 can utilize both triphosphate and diphosphate cofactors in reaction at the molecular level, but more specific with triphosphate cofactor. The NADPH is much higher than NADP+ in living cells leading to preliminary reduction action. The enzyme also showed substrate-induced inhibition unprecedented in other members of 17β-HSDs. Our previous study elucidated the structural mechanism of substrate inhibition is due to the reversely bound estrone (E1) in the substrate-binding pocket of the enzyme resulting in a dead-end complex. However, the effect of the cofactor preference on the substrate inhibition of the enzyme is not yet clear. In the present study, we solved the ternary crystal structures of 17β-HSD1 in complex with E1 and cofactor analog NAD+ . Combined with molecular dynamics simulation using the enzyme with NADH/NADPH and different oriented E1 (normally oriented, E1N; reversely oriented, E1R), such ternary structure provides a complete picture of enzyme-substrate-cofactor interactions. The results reveal that different cofactors and substrate binding mode affect the allosteric effect between the two subunits of the enzyme. And the results from MD simulations confirmed that His221 plays a key role in the formation of dead-end complex in NADPH complex, and the absence of stable interaction between His221 and E1R in the NADH complex should be the main reason for its lack of substrate inhibition.

Frustration and the Kinetic Repartitioning Mechanism of Substrate Inhibition in Enzyme Catalysis.

Substrate inhibition, whereby enzymatic activity decreases with excess substrate after reaching a maximum turnover rate, is among the most elusive phenomena in enzymatic catalysis. Here, based on a dynamic energy landscape model, we investigate the underlying mechanism by performing molecular simulations and frustration analysis for a model enzyme adenylate kinase (AdK), which catalyzes the phosphoryl transfer reaction ATP + AMP ⇋ ADP + ADP. Intriguingly, these reveal a kinetic repartitioning mechanism of substrate inhibition, whereby excess substrate AMP suppresses the population of an energetically frustrated, but kinetically activated, catalytic pathway going through a substrate (ATP)-product (ADP) cobound complex with steric incompatibility. Such a frustrated pathway plays a crucial role in facilitating the bottleneck product ADP release, and its suppression by excess substrate AMP leads to a slow down of product release and overall turnover. The simulation results directly demonstrate that substrate inhibition arises from the rate-limiting product-release step, instead of the steps for populating the catalytically competent complex as often suggested in previous works. Furthermore, there is a tight interplay between the enzyme conformational equilibrium and the extent of substrate inhibition. Mutations biasing to more closed conformations tend to enhance substrate inhibition. We also characterized the key features of single-molecule enzyme kinetics with substrate inhibition effect. We propose that the above molecular mechanism of substrate inhibition may be relevant to other multisubstrate enzymes in which product release is the bottleneck step.

Mechanism of substrate inhibition in cytochrome-c dependent NO reductases from denitrifying bacteria (cNORs)

Steady-state kinetics of cytochrome-c dependent denitrifying NO reductases (cNORs) show evidence of substrate inhibition at NO concentrations higher than 10 μM, but the mechanism of inhibition remains unclear. Here, we present low-temperature FTIR photolysis experiments carried out on the NO complex formed by addition of NO to the oxidized cNORs. A differential signal at 1261 cm−1 that downshifts with 15NO and 15N18O is assigned to a ν(NO2) from a bridging diiron-nitrito complex at the heme-nonheme diron site. Theoretical calculations reproduces observed frequencies and isotope shifts. Our experimental results confirm a prior theoretical study by Blomberg and Siegbahn [Blomberg, M. R., and Siegbahn, P. E. M. Biochemistry 2012, 51, 5173–5186] that proposed substrate inhibition through a radical combination reaction between the diferric μ-oxo group and an NO molecule to form a heme Fe(III)-nitrito-FeB(II) inhibitory complex. Stopped-flow experiments suggest that substrate inhibition also occurs after a half-reduction cycle, i.e. when fully-reduced cNOR reduces two NO molecules at the heme-nonheme diferrous active site cluster to produce one N2O molecule and the diferric cluster. These results support catalytic mechanisms that proceed through isomerization of a diferric-hyponitrite transient complex to produce a bridging diferric μ-oxo group and N2O without protonation of the putative hyponitrite intermediate.

Kinetic Modelling of Transport Inhibition by Substrates in ABC Importers

Prokaryotic ATP-binding cassette (ABC) importers require a substrate-binding protein (SBP) for the capture and delivery of the cognate substrate to the transmembrane domain (TMD) of the transporter. Various biochemical compounds have been identified that bind to the SBP but are not transported. The mechanistic basis for the “non-cognate” substrates not being transported differs. Some non-cognate substrates fail to trigger the appropriate conformational change in the SBP, resulting in loss of affinity for the TMD or the inability to allosterically activate transport. In another mechanism, the SBP cannot release the bound non-cognate substrate. Here, we used rate equations to derive the steady-state transport rate of cognate substrates of an ABC importer and investigated how non-cognate substrates influence this rate. We found that under limiting non-cognate substrate concentrations, the transport rate remains unaltered for each of the mechanisms. In contrast, at saturating substrate and SBP concentrations, the effect of the non-cognate substrate depends heavily on the respective mechanism. For instance, the transport rate becomes zero when the non-cognate substrate cannot be released by the SBP. Yet it remains unaffected when substrate release is possible but the SBP cannot dock onto the TMDs. Our work shows how the different mechanisms of substrate inhibition impact the transport kinetics, which is relevant for understanding and manipulating solute fluxes and hence the propagation of cells in nutritionally complex milieus.

Structural insights into the putative bacterial acetylcholinesterase ChoE and its substrate inhibition mechanism

Mammalian acetylcholinesterase (AChE) is well-studied, being important in both cholinergic brain synapses and the peripheral nervous systems and also a key drug target for many diseases. In contrast, little is known about the structures and molecular mechanism of prokaryotic acetylcholinesterases. We report here the structural and biochemical characterization of ChoE, a putative bacterial acetylcholinesterase from Pseudomonas aeruginosa Analysis of WT and mutant strains indicated that ChoE is indispensable for P. aeruginosa growth with acetylcholine as the sole carbon and nitrogen source. The crystal structure of ChoE at 1.35 Å resolution revealed that this enzyme adopts a typical fold of the SGNH hydrolase family. Although ChoE and eukaryotic AChEs catalyze the same reaction, their overall structures bear no similarities constituting an interesting example of convergent evolution. Among Ser-38, Asp-285, and His-288 of the catalytic triad residues, only Asp-285 was not essential for ChoE activity. Combined with kinetic analyses of WT and mutant proteins, multiple crystal structures of ChoE complexed with substrates, products, or reaction intermediate revealed the structural determinants for substrate recognition, snapshots of the various catalytic steps, and the molecular basis of substrate inhibition at high substrate concentrations. Our results indicate that substrate inhibition in ChoE is due to acetate release being blocked by the binding of a substrate molecule in a nonproductive mode. Because of the distinct overall folds and significant differences of the active site between ChoE and eukaryotic AChEs, these structures will serve as a prototype for other prokaryotic acetylcholinesterases.

Trypanosoma cruzi synthesizes proline via a Δ1-pyrroline-5-carboxylate reductase whose activity is fine-tuned by NADPH cytosolic pools.

In Trypanosoma cruzi, the etiological agent of Chagas disease, the amino acid proline participates in processes related to T. cruzi survival and infection, such as ATP production, cell differentiation, host-cell invasion, and in protection against osmotic, nutritional, and thermal stresses and oxidative imbalance. However, little is known about proline biosynthesis in this parasite. Δ1-Pyrroline-5-carboxylate reductase (P5CR, EC 1.5.1.2) catalyzes the biosynthesis of proline from Δ1-pyrroline-5-carboxylate (P5C) with concomitant NADPH oxidation. Herein, we show that unlike other eukaryotes, T. cruzi biosynthesizes proline from P5C, which is produced exclusively from glutamate. We found that TcP5CR is an NADPH-dependent cytosolic enzyme with a Kmapp for P5C of 27.7 μM and with a higher expression in the insect-resident form of the parasite. High concentrations of the co-substrate NADPH partially inhibited TcP5CR activity, prompting us to analyze multiple kinetic inhibition models. The model that best explained the obtained data included a non-competitive substrate inhibition mechanism (Kiapp=45±0.7μM). Therefore, TcP5CR is a candidate as a regulatory factor of this pathway. Finally, we show that P5C can exit trypanosomatid mitochondria in conditions that do not compromise organelle integrity. These observations, together with previously reported results, lead us to propose that in T. cruzi TcP5CR participates in a redox shuttle between the mitochondria and the cytoplasm. In this model, cytoplasmic redox equivalents from NADPH pools are transferred to the mitochondria using proline as a reduced metabolite, and shuttling to fuel electrons to the respiratory chain through proline oxidation by its cognate dehydrogenase.

Gly188Arg substitution eliminates substrate inhibition in arachidonate 11R-lipoxygenase

Lipoxygenases (LOXs) are dioxygenases that catalyze the oxygenation of polyunsaturated fatty acids to hydroperoxyl derivates. These products are precursors for different lipid mediators which are associated with pathogenesis of various diseases such as asthma, atherosclerosis and cancer. Several LOXs suffer from substrate inhibition, a potential regulatory mechanism, yet it is unclear what is the cause of this phenomenon. One such enzyme is the coral 11R-LOX which displays a significant decrease in turnover rate at arachidonic acid concentrations above 30 μM. In this report, site-directed mutagenesis and inhibition assays were employed to shed light on the mechanism of substrate inhibition in 11R-LOX. We found that introduction of a positive charge to the active site entrance with Gly188Arg substitution completely eliminates the slow-down at higher substrate concentrations. Inhibition of 11R-LOX by its catalysis product, 11(R)-hydroperoxyeicosatetraenoic acid, suggests an uncompetitive mechanism. We reason that substrate inhibition in 11R-LOX is due to additional fatty acid binding by the enzyme:substrate complex at an allosteric site situated in the very vicinity of the active site entrance.

Construction of a generalized model of the harmful substances biochemical destruction process kinetics under conditions of substrate inhibition using the methods of simulation modeling

For the purpose of obtaining the complete range of solutions for substrate inhibition of varying intensity, the mechanism of enzyme kinetics in a biocell was modeled by a multi-channel queuing system. A full range of solutions is required to make a well-grounded choice of a unified generalizing formula. The process of biodegradation with substrate inhibition was described mathematically using the method of dynamics of averages. For specific destruction rate, a full range of solutions V n of the system from minimum n =2 to limiting n →∞ order was found. It was established that the parameters of the curve shape for the solution with minimum inhibition intensity V 2 substantially stand out from the general series of the spectrum formulas. The value of the coordinate of function maximum ( n =2) V 2 is by 1.42 times higher than that of dependence ( n =3) V 3 . In the numerical experiment, the physical test was simulated by description with the help of the method of the least squares of the data, assigned by the calculation from the formulas of different structures, bearing in mind a sporadic random error. The series of numerical experiments demonstrated the capability of the formula of limiting order formula V e to describe the dependences of the whole spectrum of solutions. During describing the intermediate ratio V 3 with the help of formulas V 2 and V e , the benefit is the possible range of changing the concentrations, which is by 1.5‒2 times larger at the same relative error for dependence V е . For critical minimal order, an average relative error is sure not to exceed five percent. An increase in random error always result in statistical equality, in accuracy of describing by formulas of minimal V 2 and limiting orders V e of the data, assigned by calculation of second-order dependences. Statistical equality is achieved at the ratio of a random error to the initial error equal to ≥2.4. Collectively, the importance of the results of numerical modeling of a physical experiment involves proving the possibility of using the formula of limiting order V e as unified when describing the biodegradation processes with different mechanisms of substrate inhibition. This conclusion is proved by the adequate ( R 2 =0.9396‒0.9953) description with the help of the dependence of limiting order of experimental data on five harmful substances with varying inhibition degrees. A large amount of calculation allowed achieving a definite result – we obtained the unified formula that makes it possible to proceed to scientifically grounded design calculations for bio-treatment plants.

Crystal structures of human 17β-hydroxysteroid dehydrogenase type 1 complexed with estrone and NADP+ reveal the mechanism of substrate inhibition.

Human 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) catalyses the last step in estrogen activation and is thus involved in estrogen-dependent diseases (EDDs). Unlike other 17β-HSD members, 17β-HSD1 undergoes a significant substrate-induced inhibition that we have previously reported. Here we solved the binary and ternary crystal structures of 17β-HSD1 in complex with estrone (E1) and cofactor analog NADP+ , demonstrating critical enzyme-substrate-cofactor interactions. These complexes revealed a reversely bound E1 in 17β-HSD1 that provides the basis of the substrate inhibition, never demonstrated in estradiol complexes. Structural analysis showed that His221 is the key residue responsible for the reorganization and stabilization of the reversely bound E1, leading to the formation of a dead-end complex, which exists widely in NADP(H)-preferred enzymes for the regulation of their enzymatic activity. Further, a new inhibitor is proposed that may inhibit 17β-HSD1 through the formation of a dead-end complex. This finding indicates a simple mechanism of enzyme regulation in the physiological background and introduces a pioneer inhibitor of 17β-HSD1 based on the dead-end inhibition model for efficiently targeting EDDs. DATABASES: Coordinates and structure factors of 17β-HSD1-E1 and 17β-HSD1-E1-NADP+ have been deposited in the Protein Data Bank with accession code 6MNC and 6MNE respectively. ENZYMES: 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) EC 1.1.1.62.

The kinetic analysis of the N-methylation of 4-phenylpyridine by nicotinamide N-methyltransferase: Evidence for a novel mechanism of substrate inhibition.

The N-methylation of 4-phenylpyridine produces the neurotoxin 1-methyl-4-phenylpyridinium ion (MPP+). We investigated the kinetics of 4-phenylpyridine N-methylation by nicotinamide N-methyltransferase (NNMT) and its effect upon 4-phenylpyridine toxicity in vitro. Human recombinant NNMT possessed 4-phenylpyridine N-methyltransferase activity, with a specific activity of 1.7 ± 0.03 nmol MPP+ produced/h/mg NNMT. Although the Km for 4-phenylpyridine was similar to that reported for nicotinamide, its kcat of 9.3 × 10-5 ± 2 × 10-5 s-1 and specificity constant, kcat/Km, of 0.8 ± 0.8 s-1 M-1 were less than 0.15% of the respective values for nicotinamide, demonstrating that 4-phenylpyridine is a poor substrate for NNMT. At low (<2.5 mM) substrate concentration, 4-phenylpyridine N-methylation was competitively inhibited by dimethylsulphoxide, with a Ki of 34 ± 8 mM. At high (>2.5 mM) substrate concentration, enzyme activity followed substrate inhibition kinetics, with a Ki of 4 ± 1 mM. In silico molecular docking suggested that 4-phenylpyridine binds to the active site of NNMT in two non-redundant poses, one a substrate binding mode and the other an inhibitory mode. Finally, the expression of NNMT in the SH-SY5Y cell-line had no effect cell death, viability, ATP content or mitochondrial membrane potential. These data demonstrate that 4-phenylpyridine N-methylation by NNMT is unlikely to serve as a source of MPP+. The possibility for competitive inhibition by dimethylsulphoxide should be considered in NNMT-based drug discovery studies. The potential for 4-phenylpyridine to bind to the active site in two binding orientations using the same active site residues is a novel mechanism of substrate inhibition.

Structural Basis for the Substrate Inhibition of Proline Utilization A by Proline.

Proline utilization A (PutA) is a bifunctional flavoenzyme that catalyzes the two-step oxidation of l-proline to l-glutamate using spatially separated proline dehydrogenase (PRODH) and l-glutamate-γ-semialdehyde dehydrogenase (GSALDH) active sites. Substrate inhibition of the coupled PRODH-GSALDH reaction by proline is a common kinetic feature of PutAs, yet the structural basis for this phenomenon remains unknown. To understand the mechanism of substrate inhibition, we determined the 2.15 Å resolution crystal structure of Bradyrhizobium japonicum PutA complexed with proline. Proline was discovered in five locations remote from the PRODH active site. Most notably, strong electron density indicated that proline bound tightly to the GSAL binding site of the GSALDH active site. The pose and interactions of proline bound in this site are remarkably similar to those of the natural aldehyde substrate, GSAL, implying that proline inhibits the GSALDH reaction of PutA. Kinetic measurements show that proline is a competitive inhibitor of the PutA GSALDH reaction. Together, the structural and kinetic data show that substrate inhibition of the PutA coupled reaction is due to proline binding in the GSAL site.

Isoform-Specific Substrate Inhibition Mechanism of Human Tryptophan Hydroxylase

Tryptophan hydroxylase (TPH) catalyzes the initial and rate-limiting step in the biosynthesis of serotonin, which is associated with a variety of disorders such as depression and irritable bowel syndrome. TPH exists in two isoforms: TPH1 and TPH2. TPH1 catalyzes the initial step in the synthesis of serotonin in the peripheral tissues, while TPH2 catalyzes this step in the brain. In this study, the steady-state kinetic mechanism for the catalytic domain of human TPH1 has been determined. Varying substrate tryptophan (Trp) and tetrahydrobiopterin (BH4) results in a hybrid Ping Pong-ordered mechanism in which the reaction can either occur through a Ping Pong or a sequential mechanism depending on the concentration of tryptophan. The catalytic domain of TPH1 shares a sequence identity of 81% with TPH2. Despite the high sequence identity, differences in the kinetic parameters of the isoforms have been identified; i.e., only TPH1 displays substrate tryptophan inhibition. This study demonstrates that the difference can be traced to an active site loop which displays different properties in the TPH isoforms. Steady-state kinetic results of the isoforms, and variants with point mutations in a loop lining the active site, show that the kinetic parameters of only TPH1 are significantly changed upon mutations. Mutations in the active site loop of TPH1 result in an increase in the substrate inhibition constant, Ki, and therefore turnover rate. Molecular dynamics simulations reveal that this substrate inhibition mechanism occurs through a closure of the cosubstrate, BH4, binding pocket, which is induced by Trp binding.

Characterization of four arginine kinases in the ciliate Paramecium tetraurelia: Investigation on the substrate inhibition mechanism

The ciliate Paramecium tetraurelia contains four arginine kinase genes (AK1-4). We detected cDNA for only three of the AKs (AK1–3) via PCR. Recombinant AK1–4 were expressed in Escherichia coli and their kinetics parameters determined. AK3 showed typical substrate inhibition toward arginine, and enzymatic activity markedly decreased when arginine concentration increased. This is the first example of substrate inhibition in wild-type phosphagen kinases. To explore the substrate inhibition mechanism, site-directed mutations were generated, targeting the amino acid sequence D-D-S-Q-V at positions 77–81 in P. tetraurelia AK3. Among the mutants, substrate inhibition was lost remarkably in the S79A mutant.In spite of high amino acid sequence identity (91%) between P. tetraurelia AK3 and AK4, the enzymatic activity of AK4 was less by 3% than that of AK3. We noticed that the conservative G298 was unusually replaced by R in P. tetraurelia AK4, and we constructed two mutants, R298G/AK4 and G298R/AK3. Enzymatic activity of the former mutant was comparable with that of the wild-type AK3, whereas that of the latter mutant was dramatically reduced. Thus, we concluded that the significantly low activity of P. tetraurelia AK4 is due to the residue R298.

An excess of catalytically required motions inhibits the scavenger decapping enzyme.

The scavenger decapping enzyme hydrolyses the protecting 5′ cap structure from short mRNAs that result from exosomal degradation. Based on static crystal structures and NMR data it is apparent that the dimeric enzyme has to undergo large structural changes to bind substrate in a catalytically competent conformation. Here, we study the yeast enzyme and show that the associated opening-closing motions can be orders of magnitude faster than the catalytic turnover rate. This excess of motion is induced by binding of a second ligand to the enzyme, which occurs under high substrate concentrations. We designed a mutant that disrupts the allosteric pathway that links the second binding event to the dynamics and show that this mutant enzyme is hyperactive. Our data reveals a unique mechanism of substrate inhibition, where motions that are required for catalytic activity also inhibit efficient turnover, when they are present in excess.

Structural insight into the substrate inhibition mechanism of NADP+-dependent succinic semialdehyde dehydrogenase from Streptococcus pyogenes

Succinic semialdehyde dehydrogenases (SSADHs) are ubiquitous enzymes that catalyze the oxidation of succinic semialdehyde (SSA) to succinic acid in the presence of NAD(P)+, and play an important role in the cellular mechanisms including the detoxification of accumulated SSA or the survival in conditions of limited nutrients. Here, we report the inhibitory properties and two crystal structures of SSADH from Streptococcus pyogenes (SpSSADH) in a binary (ES) complex with SSA as the substrate and a ternary (ESS) complex with the substrate SSA and the inhibitory SSA, at 2.4 Å resolution for both structures. Analysis of the kinetic inhibitory parameters revealed significant substrate inhibition in the presence of NADP+ at concentrations of SSA higher than 0.02 mM, which exhibited complete uncompetitive substrate inhibition with the inhibition constant (Ki) value of 0.10 ± 0.02 mM. In ES-complex of SpSSADH, the SSA showed a tightly bound bent form nearby the catalytic residues, which may be caused by reduction of the cavity volume for substrate binding, compared with other SSADHs. Moreover, structural comparison of ESS-complex with a binary complex with NADP+ of SpSSADH indicated that the substrate inhibition was induced by the binding of inhibitory SSA in the cofactor-binding site, instead of NADP+. Our results provide first structure-based molecular insights into the substrate inhibition mechanism of SpSSADH as the Gram-positive bacterial SSADH.

Structure-based mutational studies of substrate inhibition of betaine aldehyde dehydrogenase BetB from Staphylococcus aureus.

Inhibition of enzyme activity by high concentrations of substrate and/or cofactor is a general phenomenon demonstrated in many enzymes, including aldehyde dehydrogenases. Here we show that the uncharacterized protein BetB (SA2613) from Staphylococcus aureus is a highly specific betaine aldehyde dehydrogenase, which exhibits substrate inhibition at concentrations of betaine aldehyde as low as 0.15 mM. In contrast, the aldehyde dehydrogenase YdcW from Escherichia coli, which is also active against betaine aldehyde, shows no inhibition by this substrate. Using the crystal structures of BetB and YdcW, we performed a structure-based mutational analysis of BetB and introduced the YdcW residues into the BetB active site. From a total of 32 mutations, those in five residues located in the substrate binding pocket (Val288, Ser290, His448, Tyr450, and Trp456) greatly reduced the substrate inhibition of BetB, whereas the double mutant protein H448F/Y450L demonstrated a complete loss of substrate inhibition. Substrate inhibition was also reduced by mutations of the semiconserved Gly234 (to Ser, Thr, or Ala) located in the BetB NAD(+) binding site, suggesting some cooperativity between the cofactor and substrate binding sites. Substrate docking analysis of the BetB and YdcW active sites revealed that the wild-type BetB can bind betaine aldehyde in both productive and nonproductive conformations, whereas only the productive binding mode can be modeled in the active sites of YdcW and the BetB mutant proteins with reduced substrate inhibition. Thus, our results suggest that the molecular mechanism of substrate inhibition of BetB is associated with the nonproductive binding of betaine aldehyde.

The mechanism of substrate inhibition in human indoleamine 2,3-dioxygenase.

Indoleamine 2,3-dioxygenase catalyzes the O2-dependent oxidation of l-tryptophan (l-Trp) to N-formylkynurenine (NFK) as part of the kynurenine pathway. Inhibition of enzyme activity at high l-Trp concentrations was first noted more than 30 years ago, but the mechanism of inhibition has not been established. Using a combination of kinetic and reduction potential measurements, we present evidence showing that inhibition of enzyme activity in human indoleamine 2,3-dioxygenase (hIDO) and a number of site-directed variants during turnover with l-tryptophan (l-Trp) can be accounted for by the sequential, ordered binding of O2 and l-Trp. Analysis of the data shows that at low concentrations of l-Trp, O2 binds first followed by the binding of l-Trp; at higher concentrations of l-Trp, the order of binding is reversed. In addition, we show that the heme reduction potential (Em0) has a regulatory role in controlling the overall rate of catalysis (and hence the extent of inhibition) because there is a quantifiable correlation between Em0 (that increases in the presence of l-Trp) and the rate constant for O2 binding. This means that the initial formation of ferric superoxide (Fe3+–O2•–) from Fe2+-O2 becomes thermodynamically less favorable as substrate binds, and we propose that it is the slowing down of this oxidation step at higher concentrations of substrate that is the origin of the inhibition. In contrast, we show that regeneration of the ferrous enzyme (and formation of NFK) in the final step of the mechanism, which formally requires reduction of the heme, is facilitated by the higher reduction potential in the substrate-bound enzyme and the two constants (kcat and Em0) are shown also to be correlated. Thus, the overall catalytic activity is balanced between the equal and opposite dependencies of the initial and final steps of the mechanism on the heme reduction potential. This tuning of the reduction potential provides a simple mechanism for regulation of the reactivity, which may be used more widely across this family of enzymes.

Improvement of l-lactic acid production by osmotic-tolerant mutant of Lactobacillus casei at high temperature

L-Lactic acid production by Lactobacillus casei was used as a model to study the mechanism of substrate inhibition and the strategy for enhancing L-lactic acid production. It was found that the concentration of cell growth and L-lactate decreased with the increase of glucose concentration and fermentation temperature. To enhance the osmotic stress resistance of the strain at high temperature, a mutant G-03 was screened and selected with 360 g/L glucose at 45°C as the selective criterion. To further increase the cell growth for lactic acid production, 3 g/L of biotin was supplemented to the medium. As a result, L: -lactate concentration by the mutant G-03 reached 198.2 g/L (productivity of 5.5 g L(-1) h(-1)) at 41°C in a 7-L fermentor with 210 g/L glucose as carbon source. L: -Lactate concentration and productivity of mutant G-03 were 115.2% and 97.8% higher than those of the parent strain, respectively. The strategy for enhancing L: -lactic acid production by increasing osmotic stress resistance at high temperature may provide an alternative approach to enhance organic acid production with other strains.

Identification and Characterization of Two Amino Acids Critical for the Substrate Inhibition of Human Dehydroepiandrosterone Sulfotransferase (SULT2A1)

Substrate inhibition is a characteristic feature of many cytosolic sulfotransferases. The differences between the complex structures of SULT2A1/DHEA and SULT2A1/PAP or SULT2A1/ADT (Protein Data Bank codes are 1J99, 1EFH, and 1OV4, respectively) have enabled us to elucidate the specific amino acids responsible for substrate inhibition. Based on the structural analyses, substitution of the smaller residue alanine for Tyr-238 (Y238A) significantly increases the K(i) value for dehydroepiandrosterone (DHEA) and totally eliminates substrate inhibition for androsterone (ADT). In addition, Met-137 was proposed to regulate the binding orientations of DHEA and ADT in SULT2A1. Complete elimination or regeneration of substrate inhibition for SULT2A1 with DHEA or ADT as substrate, respectively, was demonstrated with the mutations of Met-137 on Y238A mutant. Analysis of the Met-137 mutants and Met-137/Tyr-238 double mutants uncovered the relationship between substrate binding orientations and inhibition in SULT2A1. Our data indicate that, in the substrate inhibition mode, Tyr-238 regulates the release of bound substrate, and Met-137 controls substrate binding orientation of DHEA and ADT in SULT2A1. The proposed substrate inhibition mechanism is further confirmed by the crystal structures of SULT2A1 mutants at Met-137. We propose that both substrate binding orientations exhibited substrate inhibition. In addition, a corresponding residue in other cytosolic sulfotransferases was shown to have a function similar to that of Tyr-238 in SULT2A1.

Structural insights into substrate traffic and inhibition in acetylcholinesterase

Acetylcholinesterase (AChE) terminates nerve-impulse transmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter, acetylcholine. Substrate traffic in AChE involves at least two binding sites, the catalytic and peripheral anionic sites, which have been suggested to be allosterically related and involved in substrate inhibition. Here, we present the crystal structures of Torpedo californica AChE complexed with the substrate acetylthiocholine, the product thiocholine and a nonhydrolysable substrate analogue. These structures provide a series of static snapshots of the substrate en route to the active site and identify, for the first time, binding of substrate and product at both the peripheral and active sites. Furthermore, they provide structural insight into substrate inhibition in AChE at two different substrate concentrations. Our structural data indicate that substrate inhibition at moderate substrate concentration is due to choline exit being hindered by a substrate molecule bound at the peripheral site. At the higher concentration, substrate inhibition arises from prevention of exit of acetate due to binding of two substrate molecules within the active-site gorge.