Synthesis and SAR study of new thiazole derivatives as vascular adhesion protein-1 (VAP-1) inhibitors for the treatment of diabetic macular edema

Vascular adhesion protein-1 (VAP-1), an amine oxidase that is also known as a semicarbazide-sensitive amine oxidase (SSAO), is present in particularly high levels in human plasma, and is considered a potential therapeutic target for various inflammatory diseases, including diabetes complications such as macular edema.In our VAP-1 inhibitor program, structural modifications following high-throughput screening (HTS) of our compound library resulted in the discovery that thiazole derivative 10, which includes a guanidine group, shows potent human VAP-1 inhibitory activity (IC₅₀ of 230 nM; rat IC₅₀ of 14 nM). Moreover, compound 10 exhibited significant inhibitory effects on ocular permeability in STZ-induced diabetic rats.     

Synthetic polyamines as potential amine oxidase inhibitors: a preliminary study

In the last few decades, medicinal chemists have carried out extensive research on synthetic polyamines for use as anticancer drugs and multitarget-directed ligands in neurodegenerative diseases. The aim of this study was to evaluate the effect of some synthetic polyamines as inhibitors of two new potential targets, human semicarbazide-sensitive amine oxidase/vascular adhesion protein-1 (SSAO/VAP-1) and monoamine oxidases B (MAO B), enzymes involved in various multi-factorial diseases such as Alzheimer's disease. N,N'-Dibenzyl-dodecane-1,12-diamine (Bis-Bza-Diado), a newly synthesised compound, and ELP 12, a muscarinic cholinergic M(2) receptor antagonist, were found to behave as reversible and mixed non-competitive inhibitors of both amine oxidases (dissociation constants of about 100 μM). ELP 12 was found to be more selective for SSAO/VAP-1. Combining kinetic and structural approaches, the binding mode of ELP 12 to SSAO/VAP-1 was investigated. ELP 12 may bind at the entrance of the active site channel by ionic interactions with ASP446 and/or ASP180; one end of the polyamine may be accommodated inside the channel, reaching the TPQ cofactor area. The binding of ELP 12 induces rearrangement of the secondary structure of the enzyme and impedes substrate entry and/or product release and catalysis. These structural data reveal that the entrance and the first part of the SSAO/VAP-1 channel may be considered as a new target area, or a "secondary binding site", for modulators of human SSAO/VAP-1 activity. These results indicate ELP 12 and Bis-Bza-Diado as new "skeletons" for the development of novel SSAO/VAP-1 and MAO B inhibitors.     

A structure–activity study to identify novel and efficient substrates of the human semicarbazide-sensitive amine oxidase/VAP-1 enzyme

Kinetic studies were performed with various alkanamines as “substrate probes” of the properties of the active site of the human semicarbazide-sensitive amine oxidase/vascular adhesion protein-1 (SSAO/VAP-1). We found that the enzyme–substrate recognition step is mainly controlled by apolar interactions and that a “good” substrate has a molecular structure containing a long aliphatic chain and a second positive charge at a distance greater than 12 ? from the reactive amino group. In this context, we identified a novel substrate for the human SSAO/VAP-1, 1,12-diaminododecane (DIADO), which is characterised by the highest catalytic efficiency reported to date in comparison to the prototypic substrate benzylamine. Computational docking studies revealed the structural basis of this behaviour, highlighting the key role played by Lys393 in hindering substrate docking.     

Vascular adhesion protein-1 mediates adhesion and transmigration of lymphocytes on human hepatic endothelial cells

Vascular adhesion protein-1 (VAP-1) is an amine oxidase and adhesion receptor that is expressed by endothelium in the human liver. The hepatic sinusoids are perfused by blood at low flow rates, and sinusoidal endothelium lacks selectin expression and has low levels of CD31, suggesting that VAP-1 may play a specific role in lymphocyte recruitment to the liver. In support of this we now report the constitutive expression of VAP-1 on human hepatic sinusoidal endothelial cells (HSEC) in vitro and demonstrate that VAP-1 supports adhesion and transmigration of lymphocytes across these cells under physiological shear stress. These are the first studies to report the function of VAP-1 on primary human endothelial cells. Under static conditions lymphocyte adhesion to unstimulated HSEC was dependent on VAP-1 and ICAM-2, whereas adhesion to TNF-alpha-stimulated HSEC was dependent on ICAM-1, VCAM-1, and VAP-1. Under conditions of flow, blocking VAP-1 reduced lymphocyte adhesion to TNF-alpha-treated HSEC by 50% and significantly reduced the proportion of adherent lymphocytes that transmigrated across cytokine or LPS-activated endothelium. In addition, inhibition of the amine oxidase activity of VAP-1 reduced both adhesion and transmigration of lymphocytes to a level similar to that seen with VAP-1 Ab. Thus, VAP-1 can support transendothelial migration as well as adhesion, and both functions are dependent on its enzymatic activity. In the absence of selectins and CD31, VAP-1 may play a specific role in lymphocyte recruitment via hepatic sinusoidal endothelium. Moreover, since VAP-1 is induced on nonhepatic endothelium in response to inflammation, its ability to support lymphocyte transendothelial migration may be an important systemic function of VAP-1.     

Reaction of vascular adhesion protein-1 (VAP-1) with primary amines: mechanistic insights from isotope effects and quantitative structure-activity relationships

Human vascular adhesion protein-1 (VAP-1) is an endothelial copper-dependent amine oxidase involved in the recruitment and extravasation of leukocytes at sites of inflammation. VAP-1 is an important therapeutic target for several pathological conditions. We expressed soluble VAP-1 in HEK293 EBNA1 cells at levels suitable for detailed mechanistic studies with model substrates. Using the model substrate benzylamine, we analyzed the steady-state kinetic parameters of VAP-1 as a function of solution pH. We found two macroscopic pK(a) values that defined a bell-shaped plot of turnover number k(cat,app) as a function of pH, representing ionizable groups in the enzyme-substrate complex. The dependence of (k(cat)/K(m))(app) on pH revealed a single pK(a) value (～9) that we assigned to ionization of the amine group in free benzylamine substrate. A kinetic isotope effect (KIE) of 6 to 7.6 on (k(cat)/K(m))(app) over the pH range of 6 to 10 was observed with d(2)-benzylamine. Over the same pH range, the KIE on k(cat) was found to be close to unity. The unusual KIE values on (k(cat)/K(m))(app) were rationalized using a mechanistic scheme that includes the possibility of multiple isotopically sensitive steps. We also report the analysis of quantitative structure-activity relationships (QSAR) using para-substituted protiated and deuterated phenylethylamines. With phenylethylamines we observed a large KIE on k(cat,app) (8.01 ± 0.28 with phenylethylamine), indicating that C-H bond breakage is limiting for 2,4,5-trihydroxyphenylalanine quinone reduction. Poor correlations were observed between steady-state rate constants and QSAR parameters. We show the importance of combining KIE, QSAR, and structural studies to gain insight into the complexity of the VAP-1 steady-state mechanism.     

Adipogenesis-related increase of semicarbazide-sensitive amine oxidase and monoamine oxidase in human adipocytes

A strong induction of semicarbazide-sensitive amine oxidase (SSAO) has previously been reported during murine preadipocyte lineage differentiation but it remains unknown whether this emergence also occurs during adipogenesis in man. Our aim was to compare SSAO and monoamine oxidase (MAO) expression during in vitro differentiation of human preadipocytes and in adipose and stroma-vascular fractions of human fat depots. A human preadipocyte cell strain from a patient with Simpson-Golabi-Behmel syndrome was first used to follow amine oxidase expression during in vitro differentiation. Then, human preadipocytes isolated from subcutaneous adipose tissues were cultured under conditions promoting ex vivo adipose differentiation and tested for MAO and SSAO expression. Lastly, human adipose tissue was separated into mature adipocyte and stroma-vascular fractions for analyses of MAO and SSAO at mRNA, protein and activity levels. Both SSAO and MAO were increased from undifferentiated preadipocytes to lipid-laden cells in all the models: 3T3-F442A and 3T3-L1 murine lineages, human SGBS cell strain or human preadipocytes in primary culture. In human subcutaneous adipose tissue, the adipocyte-enriched fraction exhibited seven-fold higher amine oxidase activity and contained three- to seven-fold higher levels of mRNAs encoded by MAO-A, MAO-B, AOC3 and AOC2 genes than the stroma-vascular fraction. MAO-A and AOC3 genes accounted for the majority of their respective MAO and SSAO activities in human adipose tissue. Most of the SSAO and MAO found in adipose tissue originated from mature adipocytes. Although the mechanism and role of adipogenesis-related increase in amine oxidase expression remain to be established, the resulting elevated levels of amine oxidase activities found in human adipocytes may be of potential interest for therapeutic intervention in obesity.     

Interaction of l-lysine and soluble elastin with the semicarbazide-sensitive amine oxidase in the context of its vascular-adhesion and tissue maturation functions

The copper-containing quinoenzyme semicarbazide-sensitive amine oxidase (EC 1.4.3.21; SSAO) is a multifunctional protein. In some tissues, such as the endothelium, it also acts as vascular-adhesion protein 1 (VAP-1), which is involved in inflammatory responses and in the chemotaxis of leukocytes. Earlier work had suggested that lysine might function as a recognition molecule for SSAO/VAP-1. The present work reports the kinetics of the interaction of l-lysine and some of its derivatives with SSAO. Binding was shown to be saturable, time-dependent but reversible and to cause uncompetitive inhibition with respect to the amine substrate. It was also specific, since d-lysine, l-lysine ethyl ester and ε-acetyl-l-lysine, for example, did not bind to the enzyme. The lysine-rich protein soluble elastin bound to the enzyme relatively tightly, which may have relevance to the reported roles of SSAO in maintaining the extracellular matrix (ECM) and in the maturation of elastin. Our data show that lysyl residues are not oxidized by SSAO, but they bind tightly to the enzyme in the presence of hydrogen peroxide. This suggests that binding in vivo of SSAO to lysyl residues in physiological targets might be regulated in the presence of H₂O₂, formed during the oxidation of a physiological SSAO substrate, yet to be identified.     

Blood-brain barrier dysfunction underlying Alzheimer's disease is induced by an SSAO/VAP-1-dependent cerebrovascular activation with enhanced Aβ deposition

Dysfunctions of the vascular system directly contribute to the onset and progression of Alzheimer's disease (AD). The blood-brain barrier (BBB) shows signs of malfunction at early stages of the disease. When Abeta peptide (Aβ) is deposited on brain vessels, it induces vascular degeneration by producing reactive oxygen species and promoting inflammation. These molecular processes are also related to an excessive SSAO/VAP-1 (semicarbazide-sensitive amine oxidase) enzymatic activity, observed in plasma and in cerebrovascular tissue of AD patients. We studied the contribution of vascular SSAO/VAP-1 to the BBB dysfunction in AD using in vitro BBB models. Our results show that SSAO/VAP-1 expression is associated to endothelial activation by altering the release of pro-inflammatory and pro-angiogenic angioneurins, most highly IL-6, IL-8 and VEGF. It is also related to a BBB structure alteration, with a decrease in tight-junction proteins such as zona occludens or claudin-5. Moreover, the BBB function reveals increased permeability and leukocyte adhesion in cells expressing SSAO/VAP-1, as well as an enhancement of the vascular Aβ deposition induced by mechanisms both dependent and independent of the enzymatic activity of SSAO/VAP-1. These results reveal an interesting role of vascular SSAO/VAP-1 in BBB dysfunction related to AD progression, opening a new window in the search of alternative therapeutic targets for fighting AD.     

Towards the development of selective amine oxidase inhibitors. Mechanism-based inhibition of six copper containing amine oxidases.

Four substrate analogs, 4-(2-naphthyloxy)-2-butyn-1-amine (1), 1,4-diamino-2-chloro-2-butene (2), 1,6-diamino-2,4-hexadiyne (3), and 2-chloro-5-phthalimidopentylamine (4) have been tested as inhibitors against mammalian, plant, bacterial, and fungal copper-containing amine oxidases: bovine plasma amine oxidase (BPAO), equine plasma amine oxidase (EPAO), pea seedling amine oxidase (PSAO), Arthrobacter globiformis amine oxidase (AGAO), Escherichia coli amine oxidase (ECAO), and Pichia pastoris lysyl oxidase (PPLO). Reactions of 1,4-diamino-2-butyne with selected amine oxidases were also examined. Each substrate analog contains a functional group that chemical precedent suggests could produce mechanism-based inactivation. Striking differences in selectivity and rates of inactivation were observed. For example, between two closely related plasma enzymes, BPAO is more sensitive than EPAO to 1 and 3, while the reverse is true for 2 and 4. In general, inactivation appears to arise in some cases from TPQ cofactor modification and in other cases from alkylation of protein residues in a manner that blocks access of substrate to the active site. Notably, 1 completely inhibits AGAO at stoichiometric concentrations and is not a substrate, but is an excellent substrate of PSAO and inhibition is observed only at very high concentrations. Structural models of 1 in Schiff base linkage to the TPQ cofactor in AGAO and PSAO (for which crystal structures are available) reveal substantial differences in the degree of interaction of bound 1 with side-chain residues, consistent with the widely divergent activities. Collectively, these results suggest that the development of highly selective amine oxidase inhibitors is feasible.     

Inhibition of six copper-containing amine oxidases by the antidepressant drug tranylcypromine

Potential inhibitory effects of the clinically utilized monoamine oxidase inhibitor tranylcypromine (TCP) on mammalian, plant, bacterial, and fungal copper-containing amine oxidases have been examined. The following enzymes have been investigated: human kidney diamine oxidase (HKAO), bovine plasma amine oxidase (BPAO), equine plasma amine oxidase (EPAO), pea seedling amine oxidase (PSAO), Arthrobacter globiformis amine oxidase (AGAO), and Pichia pastoris lysyl oxidase (PPLO). Only BPAO, EPAO, and AGAO were found to lose significant levels of activity when incubated with varying amounts of TCP. Inhibition of BPAO was completely reversible, with dialysis restoring full activity. TCP inhibition of AGAO was also found to be ultimately reversible; however, dialysis did not remove all bound compounds. Chemical displacement with either substrate or a substrate analogue successfully removed all bound TCP, indicating that this compound has a high affinity for the active site of AGAO. The notable lack of TCP inhibition on HKAO argues against the inhibition of diamine oxidase as a potential source for some of the deleterious side effects occurring in patients treated with this antidepressant. The marked differences observed in behavior among these enzymes speaks to the importance of intrinsic structural differences between the active sites of copper amine oxidases (CAO) which affect reactivity with a given inhibitor.     

Vascular adhesion protein 1 (VAP-1) functions as a molecular brake during granulocyte rolling and mediates recruitment in vivo.

Granulocyte extravasation from the blood into tissues is a prerequisite for a proper inflammatory response. It is regulated by a multistep adhesion cascade consisting of successive contacts between leukocyte surface receptors and their endothelial ligands on vessels. Vascular adhesion protein 1 (VAP-1) is an endothelial surface glycoprotein with two functions. It is an enzyme (monoamine oxidase) and an adhesion molecule for lymphocytes. Its function in binding of granulocytes or in leukocyte trafficking into sites of inflammation in vivo has remained unknown. Here we show that treatment of rabbits with anti-VAP-1 monoclonal antibodies abrogates approximately 70% of granulocyte extravasation into a site of an experimental inflammation. Using intravital microscopy, VAP-1 blockade is shown to increase the velocity of the rolling granulocytes and the frequency of their jerky skippings during the rolling. In addition, the number of firmly bound leukocytes decreased by 44% when VAP-1 was rendered nonfunctional. Our results suggest that VAP-1 functions as a molecular brake early in the adhesion cascade and consequently decreases the firm adherence; it may also directly influence the transmigration step. These data elucidate a new interplayer in the granulocyte extravasation process and provide a novel physiological function for a member of the monoamine oxidase family.     

Identification of two imidazole binding sites and key residues for substrate specificity in human primary amine oxidase AOC3

Human membrane primary amine oxidase (hAOC3; also known as vascular adhesion protein-1, VAP-1) is expressed upon inflammation in most tissues, where its enzymatic activity plays a crucial role in leukocyte trafficking. We have determined two new structures of a soluble, proteolytically cleaved form of hAOC3 (sAOC3), which was extracted from human plasma. In the 2.6 ? sAOC3 structure, an imidazole molecule is hydrogen bonded to the topaquinone (TPQ) cofactor, which is in an inactive on-copper conformation, while in the 2.95 ? structure, an imidazole molecule is covalently bound to the active off-copper conformation of TPQ. A second imidazole bound by Tyr394 and Thr212 was identified in the substrate channel. We furthermore demonstrated that imidazole has an inhibitory role at high concentrations used in crystallization. A triple mutant (Met211Val/Tyr394Asn/Leu469Gly) of hAOC3 was previously reported to change substrate preferences toward those of hAOC2, another human copper-containing monoamine oxidase. We now mutated these three residues and Thr212 individually to study their distinct role in the substrate specificity of hAOC3. Using enzyme activity assays, the effect of the four single mutations was tested with four different substrates (methylamine, benzylamine, 2-phenylethylamine, and p-tyramine), and their binding modes were predicted by docking studies. As a result, Met211 and Leu469 were shown to be key residues for substrate specificity. The native structures of sAOC3 and the mutational data presented in this study will aid the design of hAOC3 specific inhibitors.     

A sedimentation procedure for the detection of binding to concanavalin A and heparin to lipoproteins.

Specific enzymatic bands in disc gel electrophoresis are generally determined by either of two methods: (i) Gel is sliced and the enzymatic activity is assayed on each slice or (ii) gel is stained histochemically, if the product of the enzymatic reaction and the dye can form an insoluble precipitate, and the activity band is located on the gel by a color band. The former is laborious and often inaccurate in the calculation of electrophoretic mobility. The latter, often nonspecific, is not applicable when the enzymatic product cannot form an insoluble precipitate with the dye. Staining with tetrazolium salt has been widely employed for amine oxidase (1–6). However, this method has limitations: (i) Tetrazolium salt is nonspecific for amine oxidase and may show artifacts (6,7), and (ii) the use of tetrazolium salt is limited only to substrates containing indolamine such as tryptamine or serotonin (8). Other substrates, like benzylamine, the most active substrate for plasma amine oxidase, do not form a color band with tetrazolium salt.This communication reports a simple spectrophotometric method for the identification of the enzymatic activity band for amine oxidase on disc gel electrophoresis. Neither slice and assay nor staining is needed. This method may possibly also be used generally for other enzyme systems which have a specific absorption at ultraviolet or visible range.     

The inhibition of semicarbazide-sensitive amine oxidase by aminohexoses

Semicarbazide-sensitive amine oxidase (EC 1.4.3.6; amine:oxygen oxidoreductase (deaminating) (copper-containing); SSAO) is a multifunctional protein. It acts under inflammatory conditions as a vascular-adhesion protein (VAP-1), mediating the adhesion of lymphocytes to vascular endothelial cells. The relationships, if any, between this adhesion function and the enzymatic functions (amine-substrate specificity and catalysis) of SSAO have not yet been defined. Since cell surface amino sugars and their derivatives are known to be involved in cell-to-cell recognition, we have investigated their possible effects on the enzyme activity of SSAO. The aminohexoses galactosamine, glucosamine and mannosamine were not oxidatively deaminated by SSAO. However, their presence during the assay of benzylamine oxidation resulted in a time-dependent inhibition. This inhibition was shown to follow saturation kinetics with respect to hexosamine concentration. Although time-dependent, the inhibition of SSAO activity was found to be reversible by dilution. In contrast, there is no such inhibition when the N-acetylamino sugar derivatives or the parent sugars (galactose, glucose and mannose) replaced the amino sugars in the reaction mixture. These results suggest that the interactions between SSAO and aminohexoses are specific and, therefore, that the cell-adhesion functions and amine-recognition functions of VAP-1/SSAO may be interlinked.     

Arylalkylamine vanadium salts as new anti-diabetic compounds

Vanadium compounds show insulin-like effects in vivo and in vitro. Several clinical studies have shown the efficacy of vanadium compounds in type 2 diabetic subjects. However, a major concern is safety, which calls for the development of more potent vanadium compounds. For that reason different laboratories develop strategies to decrease the therapeutic dose of vanadate. One of these strategies use substrates of semicarbazide-sensitive amine oxidase (SSAO)/vascular adhesion protein-1 (VAP-1), a bifunctional protein with amine oxidase activity and adhesive properties implicated in lymphocyte homing at inflammation sites. Substrates of SSAO combined with low concentrations of vanadate strongly stimulate glucose transport and GLUT4 glucose transporter recruitment to the plasma membrane in 3T3-L1 adipocytes and in rat adipocytes. This combination also shows anti-diabetic effects in various animal models of type 1 and type 2 diabetes. Benzylamine/vanadate administration generates peroxovanadium locally in pancreatic islets, which stimulates insulin secretion, and also produces peroxovanadium in adipose tissue, thereby activating glucose metabolism in adipocytes and in neighboring muscle. This opens up the possibility of using the SSAO/VAP-1 activity as a local generator of protein tyrosine phosphatase inhibitors in anti-diabetic therapy. More recently a novel class of arylalkylaminevanadium salts have shown potent insulin-mimetic effects downstream of the insulin receptor. Administration of these compounds lowers glycemia and normalizes the plasma lipid profile in type 1 and type 2 models of diabetes. The combination of different approaches to decrease vanadium doses, among them chelating agents and SSAO substrates, should permit to develop safe and efficient vanadium based agents safe for diabetes treatment.     

The pH dependence of kinetic isotope effects in monoamine oxidase A indicates stabilization of the neutral amine in the enzyme-substrate complex

A common feature of all the proposed mechanisms for monoamine oxidase is the initiation of catalysis with the deprotonated form of the amine substrate in the enzyme-substrate complex. However, recent steady-state kinetic studies on the pH dependence of monoamine oxidase led to the suggestion that it is the protonated form of the amine substrate that binds to the enzyme. To investigate this further, the pH dependence of monoamine oxidase A was characterized by both steady-state and stopped-flow techniques with protiated and deuterated substrates. For all substrates used, there is a macroscopic ionization in the enzyme-substrate complex attributed to a deprotonation event required for optimal catalysis with a pK(a) of 7.4-8.4. In stopped-flow assays, the pH dependence of the kinetic isotope effect decreases from approximately 13 to 8 with increasing pH, leading to assignment of this catalytically important deprotonation to that of the bound amine substrate. The acid limb of the bell-shaped pH profile for the rate of flavin reduction over the substrate binding constant (k(red)/K(s), reporting on ionizations in the free enzyme and/or free substrate) is due to deprotonation of the free substrate, and the alkaline limb is due to unfavourable deprotonation of an unknown group on the enzyme at high pH. The pK(a) of the free amine is above 9.3 for all substrates, and is greatly perturbed (DeltapK(a) approximately 2) on binding to the enzyme active site. This perturbation of the substrate amine pK(a) on binding to the enzyme has been observed with other amine oxidases, and likely identifies a common mechanism for increasing the effective concentration of the neutral form of the substrate in the enzyme-substrate complex, thus enabling efficient functioning of these enzymes at physiologically relevant pH.     

Cloning, sequence analysis, and characterization of the 'lysyl oxidase' from Pichia pastoris

Lysyl oxidase from Pichia pastoris has been successfully overexpressed. EPR and resonance Raman experiments have shown that copper and TPQ are present, respectively. Lysyl oxidase from P. pastoris has a similar substrate specificity to the mammalian enzyme (both have been shown to oxidize peptidyl lysine residues) and is 30% identical to the human kidney diamine oxidase (the highest of any non-mammalian source). This enzyme also has a relatively broad substrate specificity compared to other amine oxidases. Molecular modeling data suggest that the substrate channel in lysyl oxidase from P. pastoris permits greater active site access than observed in structurally-characterized amine oxidases. This larger channel may account for the diversity of substrates that are turned over by this enzyme.     

Kinetics and Yields of Pesticide Biodegradation at Low Substrate Concentrations and under Conditions Restricting Assimilable Organic Carbon

The fundamentals of growth-linked biodegradation occurring at low substrate concentrations are poorly understood. Substrate utilization kinetics and microbial growth yields are two critically important process parameters that can be influenced by low substrate concentrations. Standard biodegradation tests aimed at measuring these parameters generally ignore the ubiquitous occurrence of assimilable organic carbon (AOC) in experimental systems which can be present at concentrations exceeding the concentration of the target substrate. The occurrence of AOC effectively makes biodegradation assays conducted at low substrate concentrations mixed-substrate assays, which can have profound effects on observed substrate utilization kinetics and microbial growth yields. In this work, we introduce a novel methodology for investigating biodegradation at low concentrations by restricting AOC in our experiments. We modified an existing method designed to measure trace concentrations of AOC in water samples and applied it to systems in which pure bacterial strains were growing on pesticide substrates between 0.01 and 50 mg liter⁻¹. We simultaneously measured substrate concentrations by means of high-performance liquid chromatography with UV detection (HPLC-UV) or mass spectrometry (MS) and cell densities by means of flow cytometry. Our data demonstrate that substrate utilization kinetic parameters estimated from high-concentration experiments can be used to predict substrate utilization at low concentrations under AOC-restricted conditions. Further, restricting AOC in our experiments enabled accurate and direct measurement of microbial growth yields at environmentally relevant concentrations for the first time. These are critical measurements for evaluating the degradation potential of natural or engineered remediation systems. Our work provides novel insights into the kinetics of biodegradation processes and growth yields at low substrate concentrations.