Maximum Catalytic Rate Research Articles

Enzyme Kinetics Analysis: An online tool for analyzing enzyme initial rate data and teaching enzyme kinetics.

Enzymes are nature's catalysts, mediating chemical processes in living systems. The study of enzyme function and mechanism includes defining the maximum catalytic rate and affinity for substrate/s (among other factors), referred to as enzyme kinetics. Enzyme kinetics is a staple of biochemistry curricula and other disciplines, from molecular and cellular biology to pharmacology. However, because enzyme kinetics involves concepts rarely employed in other areas of biology, it can be challenging for students and researchers. Traditional graphical analysis was replaced by computational analysis, requiring another skill not core to many life sciences curricula. Computational analysis can be time-consuming and difficult in free software (e.g., R) or require costly software (e.g., GraphPad Prism). We present Enzyme Kinetics Analysis (EKA), a web-tool to augment teaching and learning and streamline EKA. EKA is an interactive and free tool for analyzing enzyme kinetic data and improving student learning through simulation, built using R and RStudio's ShinyApps. EKA provides kinetic models (Michaelis-Menten, Hill, simple reversible inhibition models, ternary-complex, and ping-pong) for users to fit experimental data, providing graphical results and statistics. Additionally, EKA enables users to input parameters and create data and graphs, to visualize changes to parameters (e.g., or number of measurements). This function is designed for students learning kinetics but also for researchers to design experiments. EKA (enzyme-kinetics.shinyapps.io/enzkinet_webpage/) provides a simple, interactive interface for teachers, students, and researchers to explore enzyme kinetics. It gives researchers the ability to design experiments and analyze data without specific software requirements.

Encapsulation of an enzyme-immobilized smart polymer membrane in a metal-organic framework for enhancement of catalytic performance.

Encapsulation of enzymes within porous materials has shown great promise for protecting enzymes from denaturation, increasing their tolerance to harsh environments and promoting their industrialization. However, controlling the conformational freedom of the encapsulated enzymes to enhance their catalytic performance remains a great challenge. To address this issue, herein, following immobilization of GOx and HRP on a thermo-responsive porous poly(styrene-maleic-anhydride-N-isopropylacrylamide) (PSMN) membrane, a GOx-HRP@PSMN@HZIF-8 composite was fabricated by encapsulating GOx-HRP@PSMN in hollow ZIF-8 (HZIF-8) with liposome (L) as the sacrificial template. The improved conformational freedom for enzymes arising from the hollow cavity formed in ZIF-8 through the removal of L enhanced the mass transfer and dramatically promoted the catalytic activity of the composite. Interestingly, at high temperature, the coiled PN moiety in PSMN provided the confinement effect for GOx-HRP, which also significantly boosted the catalytic performance of the composites. Compared to the maximum catalytic reaction rates (Vmax) of GOx-HRP@PSMN@LZIF-8, the free enzyme and GOx-HRP@ZIF-8, the Vmax of the GOx-HRP@PSMN@HZIF-8 composite exhibited an impressive 17.8-fold, 10.8-fold and 6.0-fold enhancement at 37 °C, respectively. The proposed composites successfully demonstrated their potential as catalytic platforms for the colorimetric detection of glucose in a cascade reaction. This study paves a new way for overcoming the current limitations of immobilizing enzymes in porous materials and the use of smart polymers for the potential fabrication of enzyme@polymer@MOF composites with tunable conformational freedom and confinement effect.

Probing conformational kinetics of catalase with and without magnetic field by single-entity collision electrochemistry

The conformational motions of enzymes are crucial for their catalytic activities, but these fluctuations are usually spontaneous and unsynchronized and thus difficult to obtain from ensemble-averaged measurements. Here, we employ label-free single-entity electrochemical measurements to monitor in real time the fluctuating enzymatic behavior of single catalase molecules toward the degradation of hydrogen peroxide. By probing the electrochemical signals of single catalase molecules at a carbon nanoelectrode, we were able to observe three distinct current traces that could be attributed to conformational changes on the sub-millisecond timescale. Whereas, nearly uniform single long peaks were observed for single catalase molecules under a moderate magnetic field due to the restricted conformational changes of catalase. By combining high-resolution current signals with a multiphysics simulation model, we studied the catalytic kinetics of catalase with and without a magnetic field, and further estimated the maximum catalytic rate and conformational transition rate. This work introduces a new complementary approach to existing single-molecule enzymology, giving further insight into the enzymatic reaction mechanism.

The importance of a charge transfer descriptor for screening potential CO2 reduction electrocatalysts

It has been over twenty years since the linear scaling of reaction intermediate adsorption energies started to coin the fields of heterogeneous and electrocatalysis as a blessing and a curse at the same time. It has established the possibility to construct activity volcano plots as a function of a single or two readily accessible adsorption energies as descriptors, but also limited the maximal catalytic conversion rate. In this work, it is found that these established adsorption energy-based descriptor spaces are not applicable to electrochemistry, because they are lacking an important additional dimension, the potential of zero charge. This extra dimension arises from the interaction of the electric double layer with reaction intermediates which does not scale with adsorption energies. At the example of the electrochemical reduction of CO2 it is shown that the addition of this descriptor breaks the scaling relations, opening up a huge chemical space that is readily accessible via potential of zero charge-based material design. The potential of zero charge also explains product selectivity trends of electrochemical CO2 reduction in close agreement with reported experimental data highlighting its importance for electrocatalyst design.

Rotary properties of hybrid F1-ATPases consisting of subunits from different species

F1-ATPase (F1) is an ATP-driven rotary motor protein ubiquitously found in many species as the catalytic portion of FoF1-ATP synthase. Despite the highly conserved amino acid sequence of the catalytic core subunits: α and β, F1 shows diversity in the maximum catalytic turnover rate Vmax and the number of rotary steps per turn. To study the design principle of F1, we prepared eight hybrid F1s composed of subunits from two of three genuine F1s: thermophilic Bacillus PS3 (TF1), bovine mitochondria (bMF1), and Paracoccus denitrificans (PdF1), differing in the Vmax and the number of rotary steps. The Vmax of the hybrids can be well fitted by a quadratic model highlighting the dominant roles of β and the couplings between α-β. Although there exist no simple rules on which subunit dominantly determines the number of steps, our findings show that the stepping behavior is characterized by the combination of all subunits.

Promotion of CO2 reduction in a nanophotocatalyst by hydrogen peroxide

Based on environmental concerns and the worldwide energy crisis, CO2 photoreduction into value-added chemicals has attracted considerable attention. However, insufficient reducing forces remain a key challenge. A nanophotocatalyst, constructed by synchronously embedding formate dehydrogenase and CdS into a zeolitic imidazolate framework, promotes CO2 photoreduction by relying on photo and chemical reducing forces. Hydrogen peroxide (H2O2) can replace water (H2O) to provide protons and electrons for CO2 photoreduction. Compared with adding reduced nicotinamide adenine dinucleotide (NADH) only, the yield of formic acid and the maximum catalytic rate increased by 2.78 and 2.55 times with adding NADH and H2O2 together as a reducing force. Protons and electrons from photolyzed H2O2 as a chemical reducing force and CdS as a photo reducing force together promote the cofactor regeneration, which in turn enhances CO2 photoreduction. This work is expected to guide a simple green method for promoting CO2 photoreduction to prepare value-added chemicals and solar fuels.

Design of enzyme@metal organic framework composites with thermo-responsivity for colourimetric detection of glucose.

Enzyme immobilization on metal-organic frameworks (MOFs) has interested researchers in recent decades due to the outstanding characteristics of MOFs. However, despite some enzyme@MOF composites exhibiting better tolerance, stability and catalysis than free enzymes, boosting the catalytic performance of stimuli-responsive polymer-grafted MOFs composites remains a challenging task. Herein, a glucose oxidase (GOx)-horseradish peroxidase (HRP)@MOF (UiO-66-NH2, U)@polymer composite with tunable catalytic ability was constructed by modification with thermo-responsive poly(N-isopropylacrylamide) (PN) via a surface-selective post-synthetic protocol. Temperature increases changed the PN-based soft armour from a "stretch" to a "coil" conformation on the MOF surface, resulting in the confinement effect and boosting the catalytic performance of the GOx-HRP@U@PN composites. Compared with its maximum catalytic reaction rate at 25 °C, the proposed composites showed 18-fold improvement in catalytic performance at 37 °C. Additionally, a colourimetric method for serum glucose analysis was developed using a GOx-HRP-based catalytic cascade reaction with a linear range from 0.1 to 2.0 mM and a low detection limit of 0.03 mM. Remarkably, the surface PN-shell-based soft armour proved to be the key factor for enhancing the catalytic performance of the as-designed composites. The co-immobilization of GOx-HRP onto the thermo-responsive U@PN surface provides a new approach for the development of highly sensitive colourimetric glucose sensing protocols.

Activity of Catalytic Ceramic Papers to Remove Soot Particles—A Study of Different Types of Soot

Diesel soot particles are of concern for both the environment and health. To catalytically remove them, it is important to know their structure and composition. There is little described in the literature on how catalysts favor the combustion of different soot fractions. In this work, programmed temperature oxidation (TPO) experiments were carried out using Co,Ce or Co,Ba,K catalysts supported on ceramic papers. Soot particles were obtained by burning diesel fuel in a vessel (LabSoot) or by filtering exhaust gases from a turbo diesel engine in a DPF filter (BenchSoot), and compared with a commercial diesel soot: Printex U. Various characterization techniques were useful to relate the characteristics of both the soot particles and the catalysts with the TPO results. The maximum catalytic soot burn rate (TM) temperatures were in the range of diesel exhaust temperatures that would facilitate in-situ regeneration of the DPF. The Co,Ba,K catalyst showed a higher catalytic effect in LabSoot, as the latter exhibited the largest primary particles and the higher order of graphene layers, for which the potassium-containing catalyst improves the contact between soot and catalyst and favors the combustion of soot, while the Co,Ce catalyst preferentially enhanced the combustion of commercial soot by supplying active oxygen.

An absorbance method for analysis of enzymatic degradation kinetics of poly(ethylene terephthalate) films

Increased interest in poly(ethylene terephthalate) (PET)-degrading enzymes (PETases) have generated efforts to find mutants with improved catalytic activity and thermostability. Here, we present a simple and fast method to determine relative enzyme kinetics through bulk absorbance measurements of released products over time. A thermostable variant of PETase from Ideonella sakaiensis was engineered (R280A S121E D186H N233C S282C) with a denaturation temperature of 69.4 ± 0.3 °C. This was used to assess the method’s ability to determine relative enzyme kinetics across variants and reveal structure–function relationships. Measurements at 24 and 72 h at 400 nM of enzyme suggest that the mutations improved catalytic rates 5- to 7-fold. On the contrary, kinetic analyses of the thermostable variant and wild-type reveal different reaction trajectories despite similar maximum catalytic rates, resulting in higher product accumulation from the thermostable variant over time. The results of the assay support the necessity for kinetic measurements to determine relationships between sequence and function for IsPETase and other PET hydrolases.

Single enzyme electroanalysis.

Traditional studies of enzymatic activity rely on the combined kinetics of millions of enzyme molecules to produce a product, an experimental approach that may wash out heterogeneities that exist between individual enzymes. Evaluating these properties on an enzyme-by-enzyme basis represents an unambiguous means of elucidating heterogeneities; however, the quantification of enzymatic activity at the single-enzyme level is fundamentally limited by the maximum catalytic rate, kcat, inherent to a given enzyme. For electrochemical methods measuring current, single enzymes must turn over greater than 107 molecules per second to produce a measurable signal on the order of 10-12 A. Enzymes with this capability are extremely rare in nature, with typical kcat values for biologically relevant enzymes falling between 1 and 10 000 s-1. Thus, clever amplification strategies are necessary to electrochemically detect the vast majority of enzymes. This review details the progress toward the electroanalytical detection and evaluation of single enzyme kinetics largely focused on the nanoimpact method, a chronoamperometric detection strategy that monitors the change in the current-time profile associated with stochastic collisions of freely diffusing entities (e.g., enzymes) onto a microelectrode or nanoelectrode surface. We discuss the experimental setups and methods developed in the last decade toward the quantification of single molecule enzymatic rates. Special emphasis is given to the limitations of measurement science in the observation of single enzyme activity and feasible methods of signal amplification with reasonable bandwidth.

Modulation of enzyme activity at nano-bio interface: A case study with acetylcholinesterase and citrate synthase adsorbed on colloidal metal nanoparticles

The effects of colloidal gold and silver nanoparticles (Au and Ag NP, respectively) were studied on the activity of acetylcholinesterase (AChE), the key enzyme in the pathology of Alzheimer's Disease (AD) and citrate synthase (CS), the mitochondrial enzyme responsible for oxidative metabolism. Being established as a primary basis of neurodegeneration, mitochondrial dysfunction connects both these two enzymes with AD. The in-vitro enzymatic activities are modulated strongly in presence of NPs. The maximum AChE catalytic rate (Vmax) increases by 30% in presence of NP systems. The Michalis-Menten constant (Km) remains unaltered in presence of AuNP; while, ~61.23% increase is noted in presence of AgNP. For the two-substrate enzyme CS, NPs were found to affect only the pathway involving oxaloacetate (OAA) as substrate. In this case, Vmax and Km increased by 21.80% and 46.60%, respectively for AuNP; while the corresponding increase were 18.20% and 33.33%, respectively for AgNP. The binding of AChE on AgNP surface occurred through random contact within very short time scale, followed by slower interaction and contact optimization within tens of nanoseconds. A series of cationic surfactants exhibited optimistic results in restoring the intrinsic activity for both the enzymes. The results highlight neurotoxicity of metal NPs in certain cases like AD and emphasize their use in pharmacological milieu only with suitable accompanying delivery medium like surfactants.

Characterization of extracellular phosphatase activities in periphytic biofilm from paddy field

Periphytic biofilms exist widely in paddy fields, but their influences on the hydrolysis of organic phosphorus (P) have rarely been investigated. In this study, a periphytic biofilm was incubated in a paddy soil solution, and hydrolysis kinetic parameters (half-saturation constant (Km) and maximum catalytic reaction rate (Vmax)), optimal environmental conditions, substrate specificity, and response to different P regimes of the phosphatase activities in the periphytic biofilm were determined, in order to characterize extracellular phosphatase activities in periphytic biofilms from paddy fields. The results indicated that the periphytic biofilm could produce an acid phosphomonoesterase (PMEase), an alkaline PMEases, and a phosphodiesterase (PDEase). These three phosphatases displayed high substrate affinity, with Km values ranging from 141.03 to 212.96 μmol L–1. The Vmax/Km ratios for the phosphatases followed the order of alkaline PMEase > acid PMEase > PDEase, which suggested that the PMEases, especially the alkaline PMEase, had higher catalytic efficiency. The optimal pH was 6.0 for the acid PMEase activity and 8.0 for the PDEase activity, and the alkaline PMEase activity increased with a pH increase from 7.0 to 12.0. The optimal temperature was 50 °C for the PMEases and 60 °C for the PDEase. The phosphatases showed high catalytic efficiency for condensed P over a wide pH range and for orthophosphate monoesters at pH 11.0, except for inositol hexakisphosphate at pH 6.0. The inorganic P supply was the main factor in the regulation of phosphatase activities. These findings demonstrated that the periphytic biofilm tested had high hydrolysis capacity for organic and condensed P, especially under P-limited conditions.

Substrate-Based Allosteric Regulation of a Homodimeric Enzyme.

Many enzymes operate through half-of-the sites reactivity wherein a single protomer is catalytically engaged at one time. In the case of the homodimeric enzyme, fluoroacetate dehalogenase, substrate binding triggers closing of a regulatory cap domain in the empty protomer, preventing substrate access to the remaining active site. However, the empty protomer serves a critical role by acquiring more disorder upon substrate binding, thereby entropically favoring the forward reaction. Empty protomer dynamics are also allosterically coupled to the bound protomer, driving conformational exchange at the active site and progress along the reaction coordinate. Here, we show that at high concentrations, a second substrate binds along the substrate-access channel of the occupied protomer, thereby dampening interprotomer dynamics and inhibiting catalysis. While a mutation (K152I) abrogates second site binding and removes inhibitory effects, it also precipitously lowers the maximum catalytic rate, implying a role for the allosteric pocket at low substrate concentrations, where only a single substrate engages the enzyme at one time. We show that this outer pocket first desolvates the substrate, whereupon it is deposited in the active site. Substrate binding to the active site then triggers the empty outer pocket to serve as an interprotomer allosteric conduit, enabling enhanced dynamics and sampling of activation states needed for catalysis. These allosteric networks and the ensuing changes resulting from second substrate binding are delineated using rigidity-based allosteric transmission theory and validated by nuclear magnetic resonance and functional studies. The results illustrate the role of dynamics along allosteric networks in facilitating function.

The global mass and average rate of rubisco

Photosynthetic carbon assimilation enables energy storage in the living world and produces most of the biomass in the biosphere. Rubisco (d-ribulose 1,5-bisphosphate carboxylase/oxygenase) is responsible for the vast majority of global carbon fixation and has been claimed to be the most abundant protein on Earth. Here we provide an updated and rigorous estimate for the total mass of Rubisco on Earth, concluding it is ≈0.7 Gt, more than an order of magnitude higher than previously thought. We find that >90% of Rubisco enzymes are found in the ≈2 × 1014 m2 of leaves of terrestrial plants, and that Rubisco accounts for ≈3% of the total mass of leaves, which we estimate at ≈30 Gt dry weight. We use our estimate for the total mass of Rubisco to derive the effective time-averaged catalytic rate of Rubisco of ≈0.03 s-1 on land and ≈0.6 s-1 in the ocean. Compared with the maximal catalytic rate observed in vitro at 25 °C, the effective rate in the wild is ≈100-fold slower on land and sevenfold slower in the ocean. The lower ambient temperature, and Rubisco not working at night, can explain most of the difference from laboratory conditions in the ocean but not on land, where quantification of many more factors on a global scale is needed. Our analysis helps sharpen the dramatic difference between laboratory and wild environments and between the terrestrial and marine environments.

Fatigue performance of ordinary concrete under discontinuous cyclic loading

Concrete structures often serves under discontinuous fatigue loading. In this work, discontinuous fatigue tests were designed for the first time to investigate the fatigue performance of ordinary concrete under discontinuous cyclic loading. During discontinuous fatigue tests, repeated stress cycles were interrupted by no-load time intervals with different durations. Results showed that time interval could promote the development of plastic deformation produced by cyclic loading. The residual strain in a spaced cycle (S cycle), which follows one time interval, was significantly larger than that in a normal cycle (N cycle), which is not preceded by any interval. The fatigue life, which was estimated by an energy dissipative model, of the specimen used in discontinuous fatigue tests was smaller than that from conventional fatigue tests. However, the dilatancy angle stayed constant in both S and N cycles. The threshold of the time interval was identified at 120 s, with the maximum catalytic rate of 117% for the tested concrete.

Comprehensive mathematical model of oxidative phosphorylation valid for physiological and pathological conditions.

We developed a mathematical model of oxidative phosphorylation (OXPHOS) that allows for a precise description of mitochondrial function with respect to the respiratory flux and the ATP production. The model reproduced flux-force relationships under various experimental conditions (state 3 and 4, uncoupling, and shortage of respiratory substrate) as well as time courses, exhibiting correct P/O ratios. The model was able to reproduce experimental threshold curves for perturbations of the respiratory chain complexes, the F1 F0 -ATP synthase, the ADP/ATP carrier, the phosphate/OH carrier, and the proton leak. Thus, the model is well suited to study complex interactions within the OXPHOS system, especially with respect to physiological adaptations or pathological modifications, influencing substrate and product affinities or maximal catalytic rates. Moreover, it could be a useful tool to study the role of OXPHOS and its capacity to compensate or enhance physiopathologies of the mitochondrial and cellular energy metabolism.

Highly Efficient Cofactors of Cu2+‐Dependent Deoxyribozymes

AbstractDeoxyribozymes can catalyze various types of reactions mostly with metal ions as their cofactors. Here we demonstrate that three copper compounds with thiosemicarbazide or semicarbazide ligands, are highly active cofactors of Cu2+‐dependent deoxyribozymes without any additional reagents. The maximum catalytic rate constant of the deoxyribozyme with dichloro(di‐thiosemicarbazide)copper as a cofactor is ∼25‐fold faster than Cu2+ cofactor under the same reaction condition. Using a variety of spectroscopies, electrochemistry, and mutagenesis, we demonstrate that both the three‐dimensional structure and redox potential of the copper center of the cofactors are essential to the catalysis of deoxyribozymes. The cofactor interacts with the enzyme‐substrate complex forming a ternary enzyme‐substrate‐cofactor complex, which is confirmed by changing the structure of the enzyme‐substrate complex through varying the temperature, or mutating the active site of the enzyme and conserved site of the substrate. The result suggests that Cu2+‐dependent deoxyribozyme requires a well‐defined active site to carry out the catalysis proficiently.

Asymmetric Reduction of Substituted 2‐Tetralones by Thermoanaerobacter pseudoethanolicus Secondary Alcohol Dehydrogenase

AbstractKetones bearing two bulky substituents, named bulky–bulky ketones, were successfully reduced to their corresponding optically enriched alcohols by using various mutants of Thermoanaerobacter pseudoethanolicus secondary alcohol dehydrogenase (TeSADH). Substituted 2‐tetralones, in particular, were reduced to 2‐tetralols with high conversion and high enantioselectivity. The pharmacological importance of substituted 2‐tetralols as key drug‐building blocks makes our biocatalytic reduction method a highly essential tool. We showed that changing the position of the substituent on the aromatic ring of 2‐tetralones impacts their binding affinity and the reaction maximum catalytic rate. Docking studies with several TeSADH mutants explain how the position of the substituent on the tetralone influences the binding orientation of substituted 2‐tetralones and their reaction stereoselectivity.

Hydrolase kinetics to detect temperature-related changes in the rates of soil organic matter decomposition

To evaluate the importance of the role of temperature on the decomposition of soil organic matter (SOM), we investigated the SOM decomposition rates and the kinetics of two hydrolases involved in carbon (C) and nitrogen (N) cycling, namely β-1,4-glucosidase (βG) and β-1,4-N-acetylglucosaminidase (NAG), and their sensitivity to temperature in representative temperate forests. Soils were collected from three spatially-separate replicate plots at study sites distributed at heights of 1233, 1060, and 825 m a.s.l. along an elevation gradient on the southern slopes of Laotuding Mountain, Northeast China. Soils were incubated at temperatures between 4 and 40 °C at 6 °C intervals for 7 days in our laboratory. Decomposition rates of SOM responded positively to temperature, and, at the same temperature, were highest in the soil from 1233 m a.s.l. than in the soils from the other elevations. SOM decomposition rates were positively correlated with the maximum activity (Vmax) of βG and the contents of total and particulate organic C, but were negatively correlated with the soil silt and clay contents. The Vmax and the Michaelis constant (Km) of the two hydrolases were positively correlated and were also correlated with increases in temperature, suggesting that the Km values could offset increases in Vmax with increases in temperature. These correlations also highlight the enzymatic tradeoff between the maximum catalytic rate and the substrate binding affinity for the two hydrolases. The catalytic efficiencies of the two hydrolases were highest at 1060 m a.s.l., followed by 1233 m a.s.l., and were lowest at 825 m a.s.l. The catalytic efficiencies were positively correlated with the soil water contents and macroaggregate contents (>250 μm), but negatively with the soil C/N ratios. The temperature sensitivities (Q10) of the SOM decomposition rates were similar at the different elevations (P > 0.05), but generally increased as the temperature increased (P < 0.05). The Q10(Vmax)/Q10(Km) values of βG and NAG increased significantly as the temperature increased from 22 to 40 °C (P < 0.001), and were generally similar between elevations (P > 0.05). Our results suggest that, in spite of the negative effects of increased temperatures on enzyme substrate affinity, increases in hydrolytic activity will lead to accelerated SOM decomposition in temperate forests.

Coordinated decline in photosynthesis and hydraulic conductance during drought stress in Zea mays

Present efforts to improve maize performance in water-limited environments are aggressively pursuing leaf-level traits, such as quantum efficiency, mesophyll osmoregulation, and stress-protein responses. However, it is possible that improvement of these traits will lead directly to hydraulic failure if hydraulics and photosynthesis are closely aligned. Here, we address the whole plant response to drought stress and ask if photosynthetic and hydraulic traits appear bundled together as a “coordinated” response, or if these traits operate largely independently of one another. Xylem conductance of leaves and stems, whole plant conductance, stomatal conductance, rate of electron transport (ETR), maximal catalytic rate of phosphoenolpyruvate carboxylase (Vpmax) (EC 4.1.1.31), and net CO2 assimilation (Amax) were measured in maize plants subjected to contrasting levels of drought stress in greenhouse and field experiments. Photosynthetic traits (Amax, ETR, Vpmax), hydraulic traits (whole plant and stem conductance) and stomatal conductance were all reduced by >80% as leaf water potentials declined below −3.0MPa. Furthermore, 83% of the variation associated with the photosynthetic and hydraulic traits measured in this study was explained by a single principal component, revealing a remarkable degree of alignment among them. Whole plant transpiration rates recovered to ca 90% of maximal values 4 d after lifting severe drought stress (Ψleaf≈−3.5MPa). Closely aligned hydraulic, photosynthetic, and stomatal responses to drought stress suggest that improvements to individual traits, in isolation to each other, may lead to the loss of plant functioning (e.g. water transport) rather than leading to marked improvements in growth.