Linear Mixed Inhibition Research Articles

Dissipation kinetics of chlorpyrifos and 3,5,6 trichloro-2-pyridinol under vegetation of different aromatic grasses: Linkage with enzyme kinetics and microbial community of soil

The dissipation of chlorpyrifos (CP) and its hydrolytic metabolite 3,5,6-trichloro-2-pyridinol (TCP) in the soil is crucial for safe agriculture. However, there is still lacking relevant information about its dissipation under different vegetation for remediation purposes. In the present study, evaluation of dissipation of CP and TCP in non-planted and planted soil with different cultivars of three types of aromatic grass viz Cymbopogon martinii (Roxb. Wats), Cymbopogon flexuosus, and Chrysopogon zizaniodes (L.) Nash was examined in light of soil enzyme kinetics, microbial communities, and root exudation. Results revealed that the dissipation of CP was well-fitted into a single first-order exponential model (SFO). A significant reduction in the half-life (DT50) of CP was observed in planted soil (30–63 days) than in non-planted soil (95 days). The presence of TCP in all soil samples was observed. The three types of the inhibitory effect of CP i.e. linear mixed inhibition (increase in enzyme-substrate affinity (Km) and decrease in enzyme pool (Vmax), un-competitive inhibition (decrease in Km and Vmax), and simple competitive inhibition were observed on soil enzymes involved in mineralization of carbon, nitrogen, phosphorus, and sulfur. The improvement in the enzyme pool (Vmax) was observed in planted soil. Streptomyces, Clostridium, Kaistobacter, Planctomyces, and Bacillus were the dominant genera in CP stress soil. CP contamination in soil demonstrated a reduction of richness in microbial diversity and enhancement of functional gene family related to cellular process, metabolism, genetic, and environmental information processing. Among all the cultivars, C. flexuosus cultivars demonstrated a higher dissipation rate of CP along with more root exudation.

Soil phosphatase assay to evaluate arsenic toxicity should be performed at the soil's actual pH

Soil phosphatase is considered an indicator to assess soil arsenic (As) pollution. In the phosphatase activity determination, a fixed buffer value (pH 5–10) is commonly used for all soils, ignoring the soil's actual pH. Here, we determined the soil phosphatase activity of 20 soils under As stress at the soils' pH, and the As inhibition mechanism was also explored by the enzyme kinetics. Our results show that soil phosphatase activity was significantly inhibited under As stress. The inhibition rate in acid soils (39.2 %) was considerably higher than in alkaline soils (25.4 %) when As concentration was 600 mg kg−1. For alkaline soils, As inhibited phosphatase by competitive inhibition or linear mixed inhibition, while for acid soils, it was more complex, including linear mixed inhibition, non-competitive inhibition, and anti-competitive inhibition. Simultaneously, our results showed that the ecological dose (ED10) described by the partial inhibition model was far below than the complete inhibition model. According to the partial inhibition model, the ED10 of As ranged from 2.66 to 164.07 mg kg−1 for alkaline soils and 0.11 to 89.95 mg kg−1 for acid soils. Moreover, Vmax/Km of phosphatase is a more sensitive index for evaluating As contamination than Vmax in partial inhibition models. The ED10 obtained based on the relationship between Vmax/Km and As concentration was 0.64–34.75 mg kg−1 for acid soils and 8.48 to 20.16 mg kg−1 for alkaline soils. This also confirms Vmax/Km as a sensitive and ideal index for assessing As pollution under soils' actual pH. Furthermore, soil pH and cation exchange capacity are dominant factors affecting As inhibition on soil phosphatase. The above kinetic studies indicate that performing the assay by adjusting the buffer pH to the soil pH is essential for more accurately evaluating arsenic toxicity.

Endogenous bioactive compounds of naked oats (Avena nuda L.) inhibit α-amylase and α-glucosidase activity

This study aims to investigate the inhibitory effects of free and bound bioactive extracts from naked oat on amylase and glucosidase activity. The free compound extract mainly included kaempferol-3-O-galactoside-6″-rhamnoside-3‴-rha, hesperetin-7-O-neohesperidoside, ginsenoside Re and soyasaponin Ba. Ten substances were detected in the bound compound extract, including ferulic acid, oxyacanthine, and trans-4-coumaric acid. The free and bound compound extracts showed dose-dependent inhibitory effects on α-amylase and α-glucosidase; the inhibition of α-amylase was reversible anti-competitive inhibition, and the inhibition of α-glucosidase was reversible linear mixed inhibition. The saponin ginsenoside Re had poor inhibitory effects on α-amylase and α-glucosidase; the saponin soyasaponin Ba had a poor inhibitory effect on α-amylase but a good inhibitory effect on α-glucosidase. After adding the extracts to the different starches, the hydrolysis rates all decreased significantly (p < 0.05). This study elucidates the low digestibility of naked oats and will help facilitate the development of low-glycemic foods using naked oats.

A Kinetic Process to Determine the Interaction Type Between Two Compounds, One of Which Is a Reaction Product, Using Alkaline Phosphatase Inhibition as a Case Study.

This study describes the development of a new methodology based on a new integrated equation which allows the determination of the kinetic parameters for two mutually non-exclusive inhibitors when one of which is produced during the time-course reaction. Alkaline phosphatase simultaneously inhibited by phosphate and urea is used to illustrate this methodology, including the evaluation of interaction effects between them. Data analyses were carried out using two integrated velocity equations: exclusive linear mixed inhibition (EMI) and non-exclusive linear mixed inhibition (NEMI). Kinetic parameters are estimated using non-linear regression and results show that (i) the interaction between enzyme and the inhibitors urea and phosphate exhibit a mutually non-exclusive behavior; (ii) more specifically, these inhibitors are non-exclusive only in free enzyme (E) species; (iii) the inhibitors also show an interaction with enzyme classified as facilitation; (iv) phosphate is a competitive inhibitor and urea a mixed inhibitor; (v) the inhibition constant for phosphate is much lower than that determined for urea. In addition, a functional Excel Spreadsheet which can be adapted to any kinetic study is also included as a supplement.

Arsenate inhibition on kinetic characteristics of alkaline phosphatase as influenced by pH

pH plays an important role on the arsenic availability to biota and enzyme activities in soil. A simulation experiment was carried out to investigate the effect of pH on the kinetic characteristics of three forms of alkaline phosphatase (free, immobilized by montmorillonite and soil) under arsenate contamination at four pH levels ranging between 7 and 10. The results showed as a function of pH, high pH favored low substrate affinity to enzyme and high maximum reaction velocity under arsenate exposure at all tested concentration ranges. Though pH could affect the kinetic parameters, the arsenate inhibition mechanisms on alkaline phosphatase (ALP) were not altered. For free ALP, a linear mixed inhibition dominated by competitive effect was displayed by arsenate. For both immobilized ALP, arsenate inhibited the enzyme activity by competing for active sites with the substrate. Based on the analysis of Michaelis constant (Km) and inhibition constant (Kic), it was shown that arsenate always exhibited stronger binding ability on enzyme than the substrate. Moreover, as the arsenate concentration increased, the ecological dose (ED10) and Kic raised in the fixed ALP, which indicates the immobilization by minerals has protected the ALP from arsenate toxicity. Our study reveals the important role of pH in the evaluation of heavy metal toxicity potentially employing soil enzyme as an indicator.

Kinetics of soil dehydrogenase in response to exogenous Cd toxicity

Soil dehydrogenase plays a role in the biological oxidation of soil organic matter and can be considered a good measure of the change of microbial oxidative activity under environmental pollutions. However, the kinetic characteristic of soil dehydrogenase under heavy metal stresses has not been investigated thoroughly. In this study, we characterized the kinetic characteristic of soil dehydrogenase in 14 soil types, and investigated how kinetic parameters changed under spiked with different concentrations of cadmium (Cd). The results showed that the Km and Vmax values of soil dehydrogenase was among 1.4–7.3mM and 15.9–235.2μMh−1 in uncontaminated soils, respectively. In latosolic red soil and brown soil, the inhibitory kinetic mechanism of Cd to soil dehydrogenase was anticompetitive inhibition with inhibition constants (Ki) of 12 and 4.7mM, respectively; in other soils belonged to linear mixed inhibition, the values of Ki were between 0.7–4.2mM. Soil total organic carbon and Ki were the major factors affecting the toxicity of Cd to dehydrogenase activity. In addition, the velocity constant (k) was more sensitive to Cd contamination compared to Vmax and Km, which was established as an early indicator of gross changes in soil microbial oxidative activity caused by Cd contamination.

Aminoalcohol-Induced Activation of Organophosphorus Hydrolase (OPH) towards Diisopropylfluorophosphate (DFP).

Aminoalcohols have been addressed as activating buffers for alkaline phosphatase. However, there is no record on the buffer activation regarding organophosphorus hydrolase (OPH). Here we reported the activating effects of aminoalcohols on OPH-catalyzed hydrolysis of diisopropylfluorophosphate (DFP), an analog molecule of G-type warfare agents. The kinetic parametors kcat, Vmax and kcat/Km in the OPH reaction were remarkably increased in the buffers (pH 8.0, 25°C) containing aminoalcohols with C2 between nitrogen (N) and oxygen (O) in their structures, including triethanolamine (TEA), diethanolamine, monoethanolamine, 1-amino-2-propanol, 2-amino-2-methyl-1-propanol, and triisopropanolamine. In contrast, much lower or no rate-enhancing effects were observed in the adding of amines, alcohols, amine/alcohol mixtures, or 3-amino-1-propanol (C3 between N and O). The 300 mM TEA further increased DFP-degrading activities of OPH mutants F132Y and L140Y, the previously reported OPH mutants with desirable activities towards DFP. However, the treatment of ethylenediaminetetraacetate (EDTA) markedly abolished the TEA-induced activation of OPH. The product fluoride effectively inhibited OPH-catalyzed hydrolysis of DFP by a linear mixed inhibition (inhibition constant Ki ~ 3.21 mM), which was partially released by TEA adding at initial or later reaction stage. The obtained results indicate the activation of OPH by aminoalcohol buffers could be attributed to the reduction of fluoride inhibition, which would be beneficial to the hydrolase-based detoxification of organophosphofluoridate.

Mechanistic analysis of allosteric and non-allosteric effects arising from nanobody binding to two epitopes of the dihydrofolate reductase of Escherichia coli

Although allosteric effector antibodies are used widely as modulators of receptors and enzymes, experimental analysis of their mechanism remains highly challenging. Here, we investigate the molecular mechanisms of allosteric and non-allosteric effector antibodies in an experimentally tractable system, consisting of single-domain antibodies (nanobodies) that target the model enzyme dihydrofolate reductase (DHFR) from Escherichia coli. A panel of thirty-five nanobodies was isolated using several strategies to increase nanobody diversity. The nanobodies exhibit a variety of effector properties, including partial inhibition, strong inhibition and stimulation of DHFR activity. Despite these diverse effector properties, chemical shift perturbation NMR epitope mapping identified only two epitope regions: epitope α is a new allosteric site that is over 10Å from the active site, while epitope β is located in the region of the Met20 loop. The structural basis for DHFR allosteric inhibition or activation upon nanobody binding to the α epitope was examined by solving the crystal structures of DHFR in complex with Nb113 (an allosteric inhibitor) and Nb179 (an allosteric activator). The structures suggest roles for conformational constraint and altered protein dynamics, but not epitope distortion, in the observed allosteric effects. The crystal structure of a β epitope region binder (ca1698) in complex with DHFR is also reported. Although CDR3 of ca1698 occupies the substrate binding site, ca1698 displays linear mixed inhibition kinetics instead of simple competitive inhibition kinetics. Two mechanisms are proposed to account for this apparent anomaly. Evidence for structural convergence of ca1698 and Nb216 during affinity maturation is also presented.

Determination of binding points of methylene blue and cationic phenoxazine dyes on human butyrylcholinesterase

In this study, the binding points of MethB and two structurally-related cationic phenoxazine dyes [meldola blue (MB) and nile blue (NB)] to human butyrylcholinesterase (BChE) were investigated by molecular docking and site directed mutagenesis. The comparative inhibitory effects of MethB, MB and NB on recombinant wild type BChE and six human BChE mutants were spectrophotometrically studied. Kinetic analyses yielded the following information: MethB and MB were found to cause nonlinear inhibition of all recombinant BChEs except Y332A, compatible with a multi-site binding model. On the other hand, MethB and MB caused linear mixed inhibition of Y332A mutant, compatible with a single binding mode. Comparing the inhibitory effects in aspect of Ki values with recombinant wild type BChE (Ki=0.042μM), MethB was found to be ∼30, 80 and 270-fold less effective as an inhibitor of Y332A, F329A and T120F, respectively. NB caused nonlinear inhibition of all recombinant BChEs. The inhibitory effect of NB on Y332A mutant was ∼370-fold lower, compared to recombinant wild type BChE (Ki=0.006μM). Considering both kinetic and molecular docking results together, it was concluded that threonine 120, phenylalanine 329 and tyrosine 332 are critical amino acids in binding of cationic phenoxazine/phenothiazine structured ligands to human BChE.

Kinetic Mechanism of Protein Arginine Methyltransferase 6 (PRMT6)

The protein arginine methyltransferases (PRMTs) are a family of enzymes that catalyze the mono- and dimethylation of arginine residues in a variety of proteins. Although these enzymes play important roles in a variety of cellular processes, aberrant PRMT activity is associated with several disease states, including heart disease and cancer. In an effort to guide the development of inhibitors targeting individual PRMTs, we initiated studies to characterize the molecular mechanisms of PRMT catalysis. Herein, we report studies on the kinetic mechanism of PRMT6. Initial velocity, product inhibition, and dead-end analog inhibition studies with the AcH4-21 and R1 peptides, as well as their monomethylated versions, indicate, in contrast to a previous report, that PRMT6 utilizes a rapid equilibrium random mechanism with dead-end EAP and EBQ complexes.

Monitoring Enzyme Reaction and Screening of Inhibitors of Acetylcholinesterase by Quantitative Matrix-Assisted Laser Desorption/Ionization Fourier Transform Mass Spectrometry

A matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS)-based assay was developed for kinetic measurements and inhibitor screening of acetylcholinesterase. Here, FTMS coupled to MALDI was applied to quantitative analysis of choline using the ratio of choline/acetylcholine without the use of additional internal standard, which simplified the experiment. The Michaelis constant (K(m)) of acetylcholinesterase (AChE) was determined to be 73.9 micromol L(-1) by this approach. For Huperzine A, the linear mixed inhibition of AChE reflected the presence of competitive and noncompetitive components. The half maximal inhibitory concentration (IC(50)) value of galantamine obtained for AChE was 2.39 micromol L(-1). Inhibitory potentials of Rhizoma Coptidis extracts were identified with the present method. In light of the results the referred extracts as a whole showed inhibitory action against AChE. The use of high-resolution FTMS largely eliminated the interference with the determination of ACh and Ch, produced by the low-mass compounds of chemical libraries for inhibitor screening. The excellent correlation with the reported kinetic parameters confirms that the MS-based assay is both accurate and precise for determining kinetic constants and for identifying enzyme inhibitors. The obvious advantages were demonstrated for quantitative analysis and also high-throughput characterization. This study offers a perspective into the utility of MALDI-FTMS as an alternate quantitative tool for inhibitor screening of AChE.

Bone Sialoprotein Binding to Matrix Metalloproteinase-2 Alters Enzyme Inhibition Kinetics

Bone sialoprotein (BSP) is a secreted glycophosphoprotein normally restricted in expression to skeletal tissue that is also induced by multiple neoplasms in vivo. Previous work has shown that BSP can bind to matrix metalloproteinase-2 (MMP-2). Because of MMP-2 activity in promoting tumor progression, potential therapeutic inhibitors were developed, but clinical trials have been disappointing. The effect of BSP on MMP-2 modulation by inhibitors was determined with purified components and in cell culture. Enzyme inhibition kinetics were studied using a low-molecular weight freely diffusable substrate and purified MMP-2, BSP, and natural (tissue inhibitor of matrix metalloproteinase-2) and synthetic (ilomastat and oleoyl- N-hydroxylamide) inhibitors. We determined parameters of enzyme kinetics by varying substrate concentrations at different fixed inhibitor concentrations added to MMP-2 alone, MMP-2 and BSP, or preformed MMP-2-BSP complexes and solving a general linear mixed inhibition rate equation with a global curve fitting program. Two in vitro angiogenesis model systems employing human umbilical vein endothelial cells (HUVECs) were used to follow BSP modulation of MMP-2 inhibition and tubule formation. The presence of BSP increased the competitive K I values between 15- and 47-fold for natural and synthetic inhibitors. The extent of tubule formation by HUVECs cocultured with dermal fibroblasts was reduced in the presence of inhibitors, while the addition of BSP restored vessel formation. A second HUVEC culture system demonstrated that tubule formation by cells expressing BSP could be inhibited by an activity blocking antibody against MMP-2. BSP modulation of MMP-2 activity and inhibition may define its biological role in promoting tumor progression.

Modelling the effect of superatmospheric oxygen concentrations on in vitro mushroom PPO activity

AbstractThe kinetics of polyphenol oxidase (PPO, EC 1.14.18.1) with respect to oxygen concentrations from 5 to 100% using chlorogenic acid (CGA) as substrate was examined. In vitro mushroom PPO activity was determined by measuring the consumption of oxygen during the oxidation reaction. A differential Michaelis–Menten model was fitted to the obtained total depletion curves. The product concentration as well as the concentration of oxygen had a clear inhibitory effect on the reaction rate. However, the inhibitory effect of oxygen was more evident at low product concentration. A linear mixed inhibition model that considered both the product (oxidised CGA) and oxygen as inhibitors was developed. A model with the product as a competitive inhibitor and oxygen as an uncompetitive inhibitor was the most appropriate to explain the reaction kinetics. The values of the inhibition constants calculated from the model were 0.0032 mmol L−1 for Km (Michaelis–Menten constant related to oxygen), 0.023 mmol L−1 for Kmc (constant for competitive inhibition due to the product), 1.630 mmol L−1 for Kmu (constant for uncompetitive inhibition due to oxygen) and 1.77 × 10−4 mmol L−1 s−1 for Vmax (maximum reaction rate). The results indicate that superatmospheric oxygen concentrations could be effective in preventing enzymatic browning by PPO. Copyright © 2006 Society of Chemical Industry

Inhibition of human plasma cholinesterase by malachite green and related triarylmethane dyes: Mechanistic implications

The inhibitory effects of the cationic triarylmethane (TAM +) dyes, pararosaniline (PR +), malachite green (MG +), and methyl green (MeG +) on human plasma cholinesterase (BChE) were studied at 25 °C in 100 mM Mops, pH 8.0, with butyrylthiocholine as substrate. PR + and MG + caused linear mixed inhibition of enzyme activity. The respective inhibitory parameters were K i = 1.9 ± 0.23 μM, α = 13 ± 48, β = 0 and K i = 0.28 ± 0.037 μM, α = 23 ± 7.4, β = 0. MeG + acted as a competitive inhibitor with K i = 0.12 ± 0.017 μM ( α, ∞, β, not applicable). The K i values were within the same range reported for a number of ChE inhibitors including propidium ion, donepezil, and the phenothiazines, suggesting that TAM +s are active site ligands. On the other hand, the α values failed to correlate with values previously reported for a number of ChE inhibitors. It appears that mixed inhibition is the combined result of more than one type of binding and S–I interference. The impact of ligands at the choline-specific and peripheral anionic sites (or, possibly, accessory structural domains) on BChE activity needs to be studied in further detail.

Linear competitive inhibition of human tissue kallikrein by 4-aminobenzamidine and benzamidine and linear mixed inhibition by 4-nitroaniline and aniline.

Hydrolysis of D-valyl-L-leucyl-L-arginine p-nitroanilide (7.5-90.0 microM) by human tissue kallikrein (hK1) (4.58-5.27 nM) at pH 9.0 and 37 degrees C was studied in the absence and in the presence of increasing concentrations of 4-aminobenzamidine (96-576 microM), benzamidine (1.27-7.62 mM), 4-nitroaniline (16.5-66 microM) and aniline (20-50 mM). The kinetic parameters determined in the absence of inhibitors were: Km = 12.0 +/- 0.8 microM and k cat = 48.4 +/- 1.0 min(-1). The data indicate that the inhibition of hK1 by 4-aminobenzamidine and benzamidine is linear competitive, while the inhibition by 4-nitroaniline and aniline is linear mixed, with the inhibitor being able to bind both to the free enzyme with a dissociation constant Ki yielding an EI complex, and to the ES complex with a dissociation constant Ki', yielding an ESI complex. The calculated Ki values for 4-aminobenzamidine, benzamidine, 4-nitroaniline and aniline were 146 +/- 10, 1,098 +/- 91, 38.6 +/- 5.2 and 37,340 +/- 5,400 microM, respectively. The calculated Ki' values for 4-nitroaniline and aniline were 289.3 +/- 92.8 and 310,500 +/- 38,600 microM, respectively. The fact that Ki'>Ki indicates that 4-nitroaniline and aniline bind to a second binding site in the enzyme with lower affinity than they bind to the active site. The data about the inhibition of hK1 by 4-aminobenzamidine and benzamidine help to explain previous observations that esters, anilides or chloromethyl ketone derivatives of Nalpha-substituted arginine are more sensitive substrates or inhibitors of hK1 than the corresponding lysine compounds.

Combination plots as graphical tools in the study of enzyme inhibition.

Although statistical regression has become the method of choice in the analysis of enzyme kinetics, graphical methods continue to be useful on account of their illustrative capabilities. It is pointed out in this paper that enzyme inhibition data may be presented more efficiently as a single linear plot than the traditional way as a family of lines. This approach has been taken previously by Hunter and Downs [Hunter and Downs (1945) J. Biol. Chem. 157, 427-446] but has remained neglected. A new version of this type of plot (combination plot) has been devised which is linear for competitive, non-competitive, uncompetitive and linear mixed inhibition and has a characteristic appearance for each type of inhibition behaviour. The slopes and intercepts not only indicate directly the dissociation constant but also provide quantitative criteria for the nature of inhibition. This plot should serve as a useful graphical tool in enzyme research as well as in biochemical education.

Mixed-type inhibition of bovine lung angiotensin converting enzyme by lisinopril and its dansyl derivative

The steady-state inhibition of bovine lung angiotensin converting enzyme (ACE; EC 3.4.15.1) by the slow-binding inhibitor lisinopril and its dansyi derivative conformed to a linear mixed inhibition model with inhibitor binding to ES as well as to E. Studied at pH 8, 35°, and using N-(3-[2-furyl]-acryloyl)phe-gly-gly as substrate, the approach to steady-state activity at different substrate concentrations pointed to slow isomerizations in both El and EIS. While an inhibitory scheme involving a single I-binding site adequately accounts for the data presented, information relating to the primary structure of ACE brings up a two-site alternative which remains to be tested.

Ligand exclusion on acetylcholinesterase

This paper examines covalent reactivity of AchE with respect to cationic and uncharged methylphosphonates and substrates in the absence and presence of cationic ligands selective for the active center and the peripheral anionic site. The organophosphorus inhibitors are enantiomeric alkyl methylphosphonothioates (1-5) containing cycloheptyl and isopropyl phosphono ester groups and S-methyl, S-n-pentyl, and S-[beta-(trimethylammonio)ethyl] leaving groups; these agents differ in their configuration about phosphorus and their steric, hydrophobic, and electrostatic characteristics. The synthetic substrates examined are acetylthiocholine, p-nitrophenyl acetate, and 7-acetoxy-4-methylcoumarin (7AMC). Antagonism of the methylphosphonothioate reaction by cationic ligands is strongly dependent on the nature of both the cation and the methylphosphonate but independent of the configuration about phosphorus. While all cations cause linear mixed inhibition of acetylthiocholine hydrolysis, there are observed a variety of inhibition patterns of 7AMC and p-nitrophenyl acetate hydrolysis that are distinctly nonlinear, as well as patterns in which the reciprocal plots intersect in the upper right quadrant. Strong antagonism of cationic (methylphosphonyl)thiocholines correlates very well with linear inhibition of acetylthiocholine. Ligands that cause only negligible antagonism of the uncharged methylphosphonates display nonlinear inhibition of uncharged substrates. These relationships, since they are most pronounced for peripheral site ligands and are strongly dependent on the charge carried by the reactant, suggest that the peripheral anionic site alters enzyme reactivity through an electrostatic interaction with the net negative active center. Such behavior indicates a potential role for the peripheral anionic site in conserving AchE catalytic efficiency within a narrow range of values.

Inhibition of equine S-adenohomocysteine hydrolase by 2'-deoxyadenosine

1. 1. 2'-Deoxyadenosine and 9-β-D-arabinofuranosyladenine (ARA) are apparent suicide inhibitors for equine S-adenosylhomocysteine hydrolase. 2. 2. In initial velocity studies of the synthetic reaction converting adenosine and homocysteine to S-adenosylhomocysteine, adenine, adenosine 5'-triphosphate, and 9-β- d-arabinofuranosyladenine were found to be competitive inhibitors with Kf s of 3.8μM, 1.1 mM, and 30μM, respectively. 3. 3. In contrast, linear mixed inhibition was observed for 2'-deoxyadenosine, indicating that 2'-deoxyadenosine must bind in more than one fashion to the enzyme.