Steady-state Enzyme Kinetics Research Articles

A flexible linker of 8-amino acids between the membrane binding segment and the FMN domain of cytochrome P450 reductase is necessary for optimal activity

The diflavin NADPH-cytochrome P450 reductase (CYPOR) plays a critical role in human cytochrome P450 (CYP) activity by sequentially delivering two electrons from NADPH to CYP enzymes during catalysis. Although electron transfer to forty-eight human CYP enzymes by the FMN hydroquinone of CYPOR is well-known, the role of the linker between the NH2-terminus membrane-binding domain (MBD) and FMN domain in supporting the activity of P450 enzymes remains poorly understood. Here we demonstrate that a linker with at least eight residues is required to form a functional CYPOR-CYP2B4 complex. The linker has been shortened in two amino-acid increments from Phe44 to Ile57 using site directed mutagenesis. The ability of the deletion mutants to support cytochrome P450 2B4 (CYP2B4) catalysis and reduce ferric CYP2B4 was determined using an in vitro assay and stopped-flow spectrophotometry. Steady-state enzyme kinetics showed that shortening the linker by 8–14 amino acids inhibited (63–99%) the ability of CYPOR to support CYP2B4 activity and significantly increased the Km of CYPOR for CYP2B4. In addition, the reductase mutants decreased the rate of reduction of ferric CYP2B4 (46–95%) compared to wildtype when the linker was shortened by 8–14 residues. These results indicate that a linker with a minimum length of eight residues is necessary to enable the FMN domain of reductase to interact with CYP2B4 to form a catalytically competent complex. Our study provides evidence that the length of the MBD-FMN domain linker is a major determinant of the ability of CYPOR to support CYP catalysis and drug metabolism by P450 enzymes. PreambleThis manuscript is dedicated in memory of Dr. James R. Kincaid who was the doctoral advisor to Dr. Freeborn Rwere and a longtime collaborator and friend of Dr. Lucy Waskell. Dr. James R. Kincaid was a distinguished professor of chemistry specializing in resonance Raman (rR) studies of heme proteins. He inspired Dr. Rwere (a Zimbabwean native) and three other Zimbabweans (Dr. Remigio Usai, Dr. Daniel Kaluka and Ms. Munyaradzi E. Manyumwa) to use lasers to document subtle changes occurring at heme active site of globin proteins (myoglobin and hemoglobin) and cytochrome P450 enzymes. Dr. Rwere appreciate his contributions to the development of talented Black scientists from Africa.

Steady-state kinetic analysis of reversible enzyme inhibitors: A case study on calf intestine alkaline phosphatase.

Enzymes are important drug targets and inhibition of enzymatic activity is an important therapeutic strategy. Enzyme assays measuring catalytic activity are utilized in both the discovery and development of new drugs. Colorimetric assays based on the release of 4-nitrophenol from substrates are commonly used. 4-Nitrophenol is only partly ionized to 4-nitrophenolate under typical assay conditions (pH 7-9) leading to under-estimation of product formation rates due to the much lower extinction coefficient of 4-nitrophenol compared to 4-nitrophenolate. Determination of 4-nitrophenol pKa values based on absorbance at 405nm as a function of experimental pH values is reported, allowing for calculation of a corrected extinction coefficient at the assay pH. Characterization of inhibitor properties using steady-state enzyme kinetics is demonstrated using calf intestine alkaline phosphatase and 4-nitrophenyl phosphate as substrate at pH ∼8.2. The following kinetic parameters were determined: Km=40±3µM; Vmax=72.8±1.2µmolmin-1mg protein-1; kcat=9.70±0.16s-1; kcat/Km=2.44±0.16 × 105 M-1s-1 (mean±SEM, N=4). Sodium orthovanadate and EDTA were used as model inhibitors and the following pIC50 values were measured using dose-response curves: 6.61±0.08 and 3.07±0.03 (mean±SEM, N=4). Rapid dilution experiments determined that inhibition was reversible for sodium orthovanadate and irreversible for EDTA. A Ki value for orthovanadate of 51±8nM (mean±SEM, N=3) was determined. Finally, data analysis and statistical design of experiments are discussed.

High-Throughput Steady-State Enzyme Kinetics Measured in a Parallel Droplet Generation and Absorbance Detection Platform.

Microfluidic water-in-oil emulsion droplets are becoming a mainstay of experimental biology, where they replace the classical test tube. In most applications, such as ultrahigh-throughput directed evolution, the droplet content is identical for all compartmentalized assay reactions. When emulsion droplets are used for kinetics or other functional assays, though, concentration dependencies of initial rates that define Michaelis-Menten parameters are required. Droplet-on-demand systems satisfy this need, but extracting large amounts of data is challenging. Here, we introduce a multiplexed droplet absorbance detector, which─coupled to semi-automated droplet generation─forms a tubing-based droplet-on-demand system able to generate and extract quantitative datasets from defined concentration gradients across multiple series of droplets for multiple time points. The emergence of a product is detected by reading the absorbance of the droplet sets at multiple, adjustable time points by reversing the flow direction after each detection, so that the droplets pass a line scan camera multiple times. Detection multiplexing allows absorbance values at 12 distinct positions to be measured, and enzyme kinetics are recorded for label-free concentration gradients that are composed of about 60 droplets each, covering as many concentrations. With a throughput of around 8640 data points per hour, a 10-fold improvement compared to the previously reported single point detection method is achieved. In a single experiment, 12 full datasets of high-resolution and high-accuracy Michaelis-Menten kinetics were determined to demonstrate the potential for enzyme characterization for glycosidase substrates covering a range in enzymatic hydrolysis of 7 orders of magnitude in kcat/KM. The straightforward setup, high throughput, excellent data quality, and wide dynamic range that allows coverage of diverse activities suggest that this system may serve as a miniaturized spectrophotometer for detailed analysis of clones emerging from large-scale combinatorial experiments.

Photoswitching of Feedback Inhibition by Tryptophan in Anthranilate Synthase.

The artificial regulation of enzymatic activity by light is an important goal of synthetic biology that can be achieved by the incorporation of light-responsive noncanonical amino acids via genetic code expansion. Here, we apply this concept to anthranilate synthase from Salmonella typhimurium (stTrpE). This enzyme catalyzes the first step of tryptophan biosynthesis, and its activity is feedback-inhibited by the binding of the end-product of the pathway to an allosteric site. To put this feedback inhibition of stTrpE by tryptophan under the control of light, we individually replaced 15 different amino acid residues with the photosensitive noncanonical amino acid o-nitrobenzyl-O-tyrosine (ONBY). ONBY contains a sterically demanding caging group that was meant to cover the allosteric site. Steady-state enzyme kinetics showed that the negative effect of tryptophan on the catalytic activity of the two variants stTrpE-K50ONBY and stTrpE-Y455ONBY was diminished compared to the wild-type enzyme by 1 to 2 orders of magnitude. Upon light-induced decaging of ONBY to the less space-consuming tyrosine residue, tryptophan binding to the allosteric site was restored and catalytic activity was inhibited almost as efficiently as observed for wild-type stTrpE. Based on these results, direct photocontrol of feedback inhibition of stTrpE-K50ONBY and stTrpE-Y455ONBY could be achieved by irradiation during the reaction. Molecular modeling studies allowed us to rationalize the observed functional conversion from the noninhibited caged to the tryptophan-inhibited decaged states. Our study shows that feedback inhibition, which is an important mechanism to regulate key metabolic enzymes, can be efficiently controlled by the purposeful use of light-responsive noncanonical amino acids.

Risedronate and Methotrexate Are High-Affinity Inhibitors of New Delhi Metallo-β-Lactamase-1 (NDM-1): A Drug Repurposing Approach.

Bacteria expressing New Delhi metallo-β-lactamase-1 (NDM-1) can hydrolyze β-lactam antibiotics (penicillins, cephalosporins, and carbapenems) and, thus, mediate multidrug resistance. The worldwide dissemination of NDM-1 poses a serious threat to public health, imposing a huge economic burden in the development of new antibiotics. Thus, there is an urgent need for the identification of novel NDM-1 inhibitors from a pool of already-known drug molecules. Here, we screened a library of FDA-approved drugs to identify novel non-β-lactam ring-containing inhibitors of NDM-1 by applying computational as well as in vitro experimental approaches. Different steps of high-throughput virtual screening, molecular docking, molecular dynamics simulation, and enzyme kinetics were performed to identify risedronate and methotrexate as the inhibitors with the most potential. The molecular mechanics/generalized Born surface area (MM/GBSA) and molecular dynamics (MD) simulations showed that both of the compounds (risedronate and methotrexate) formed a stable complex with NDM-1. Furthermore, analyses of the binding pose revealed that risedronate formed two hydrogen bonds and three electrostatic interactions with the catalytic residues of NDM-1. Similarly, methotrexate formed four hydrogen bonds and one electrostatic interaction with NDM-1’s active site residues. The docking scores of risedronate and methotrexate for NDM-1 were –10.543 kcal mol−1 and −10.189 kcal mol−1, respectively. Steady-state enzyme kinetics in the presence of risedronate and methotrexate showed a decreased catalytic efficiency (i.e., kcat/Km) of NDM-1 on various antibiotics, owing to poor catalytic proficiency and affinity. The results were further validated by determining the MICs of imipenem and meropenem in the presence of risedronate and methotrexate. The IC50 values of the identified inhibitors were in the micromolar range. The findings of this study should be helpful in further characterizing the potential of risedronate and methotrexate to treat bacterial infections.

1390. Durlobactam, a Diazabicyclooctane (DBO) β-lactamase Inhibitor (BLI), Inhibits BlaC and Peptidoglycan (PG) Transpeptidases of Mycobacterium tuberculosis (Mtb): A Novel Approach to Therapeutics for Tuberculosis (TB)?

BackgroundNovel therapies for multidrug-resistant TB are needed and new BLIs could answer this call. Mtb encodes for BlaC, a class A β-lactamase. BlaC is inhibited by clavulanate (CLA) while the DBO avibactam (AVI) is an inefficient inhibitor (low k2/K value). Carbapenems are hydrolyzed slowly by BlaC (low kcat/Km value) making them “dual action” compounds that inhibit both BlaC and PG transpeptidases, the intended β-lactam targets. DBOs inhibit PG transpeptidases in other bacteria. To explore the therapeutic potential of new DBOs against Mtb, we compared the inhibitor activity of AVI, relebactam (REL), and durlobactam (DUR, formerly ETX2514) against BlaC and Mtb PG transpeptidases using a biochemical approach. We also investigated the ability of DUR to lower minimum inhibitory concentrations (MICs) of β-lactams against Mtb H37Rv. MethodsMass spectrometry was performed to capture acyl-enzyme complexes (AECs) of purified BlaC and PG transpeptidases (PonA1, LdtMt1, LdtMt2, LdtMt3, and LdtMt5) with β-lactams and BLIs. Steady-state enzyme kinetics were determined using nitrocefin as a substrate. MICs with amoxicillin (AMX), meropenem (MER), CLA, and DUR alone and in combination against Mtb H37Rv were assessed using a microdilution method.ResultsDUR alone had a MIC of 2 µg/mL with Mtb H37Rv (Table 1). BlaC formed AECs with all carbapenems and BLIs. BlaC had lower Ki app and higher k2/K with DUR than those with AVI and REL and comparable to those with CLA; however, with a period of pre-incubation, AVI fully inhibits BlaC (Table 2). The carbapenems and DUR formed the most AECs with PG transpeptidases of the β-lactams and BLIs respectively; PG transpeptidases had lower Ki app values with DUR than those with AVI (Table 3). Table 1. Minimum Inhibitory Concetrations for Mycobacterium tuberculosis H37Rv ConclusionDUR alone has some antimicrobial activity against Mtb H37Rv. The likely mechanism that underlies this activity is inhibition of BlaC and several PG transpeptidases. Inhibition of enzyme targets with DUR was more potent and efficient than AVI and REL. DUR in combination with β-lactams lowered MICs but the DUR concentration used was higher than its MIC. Our findings support the exploration of novel BLIs against BlaC and PG transpeptidases with the ultimate goal of repurposing these drugs for the treatment of TB. Disclosures Robert A. Bonomo, MD, entasis (Research Grant or Support)Merck (Grant/Research Support)NIH (Grant/Research Support)VA Merit Award (Grant/Research Support)VenatoRx (Grant/Research Support)

1286. Taniborbactam Inhibits Cefepime-Hydrolyzing Variants of Pseudomonas-derived Cephalosporinase (PDC)

BackgroundPDC is a class C β-lactamase in P. aeruginosa. PDC-88 is a variant characterized by a Thr-Pro amino acid deletion in the R2-loop (Δ289-290; Fig. 1). This deletion reduces susceptibility to cefepime (FEP), ceftazidime (CAZ), and ceftolozane-tazobactam (TOL/TZB), but the mechanism for this “gain of function” is unknown. Taniborbactam (TAN) is a novel cyclic boronate β-lactamase inhibitor (BLI) with activity against all four β-lactamase classes and is currently undergoing a phase 3 clinical trial paired with FEP. Herein, we studied the extended-spectrum AmpC (ESAC) phenotype of PDC-88 and examined the ability of TAN to inhibit this variant. Structure of PDC-1 (PDB ID: 4GZB) with PDC-88 deleted residues in red and substitutions in green. All four amino acid substitutions (T79A, V178L, V329I, and G346A) are common (occurring in 10% or more of PDC variants) and have not been associated with resistance. Image rendered using UCSF Chimera.MethodsBroth microdilution minimum inhibitory concentrations (MIC) were determined in accordance with CLSI. PDC-3 and PDC-88 were purified, and steady-state enzyme kinetics were determined. Quadrupole time-of-flight mass spectrometry (Q-TOF-MS) was performed.ResultsIn isogenic E. coli expressing PDC-3 or PDC-88, FEP MIC increased 8- or 128-fold, respectively, compared to the empty vector. Addition of TAN at 4 μg/ml restored FEP activity with MIC lowered to 0.25 μg/ml (Table 1) for both PDC-3 and PDC-88 bearing strains. PDC-88 demonstrated a 9-fold lower KM, 3.4-fold lower kcat, and 2.6-fold higher kcat/KM for FEP compared to PDC-3 (Table 2A). TAN Ki values were 4- to 10-fold lower than avibactam (AVI) and 40- to 95-fold lower than TZB. The TAN acylation constant (k2/K) was 7- to 12-fold greater than AVI and 133- to 366-fold higher than TZB (Table 2B). Q-TOF-MS revealed faster deacylation of FEP by PDC-88 compared to TOL and CAZ. TOL was acylated and deacylated by PDC-88 but not by PDC-3. CAZ was readily acylated but slowly deacylated by PDC-88 compared to PDC-3 (Fig. 2). Cefepime Minimum Inhibitory Concentration (MIC) for PDC-1 and a series of partial R2-loop deletions with and without taniborbactam, avibactam, and tazobactam. In all variants, taniborbactam and avibactam restored susceptiblity while tazobactam is less effective against PDC-88 and variants. Summary of kinetic constants. (A) Comparison of Michaelis constant (KM), turnover number (kcat), and catalytic efficiency (kcat/KM) of nitrocefin and cefepime with PDC-3 and PDC-88. (B) Comparison of inhibition constant (Ki) and acylation constant (k2/K) for avibactam, tazobactam, and taniborbactam with PDC-3 and PDC-88. Graphical summary of mass spectrometry results for substrate acyl-enzyme complex capture experiments. FEP, cefepime; CAZ, ceftazidime; TOL, ceftolozane. Primes indicate a modified substrate (loss of R2 group). TOL does not form an acyl-enzyme complex with PDC-3.ConclusionDifferent kinetic constants are responsible for the elevated cephalosporin MICs. We posit that PDC-88 increases FEP MIC by enhanced hydrolysis; TOL MICs by enabling acylation; and CAZ MICs by both trapping and enhanced hydrolysis. TAN inhibits both PDC-3 and PDC-88 with similar kinetic profiles. Notably, TAN appears to be a more efficient inhibitor of PDC than current BLIs targeted for use against P. aeruginosa (lower Ki, higher k2/K values). The combination of TAN and FEP may represent an important treatment option for P. aeruginosa isolates that develop ESAC phenotypes.Disclosures Focco van den Akker, PhD, Venatorx Pharmaceuticals, Inc. (Grant/Research Support) Brittany A. Miller, BS, Venatorx Pharmaceuticals, Inc. (Employee) Tsuyoshi Uehara, PhD, Venatorx Pharmaceuticals, Inc. (Employee) David A. Six, PhD, Venatorx Pharmaceuticals, Inc. (Employee) Krisztina M. Papp-Wallace, Ph.D., Merck & Co., Inc. (Grant/Research Support)Spero Therapeutics, Inc. (Grant/Research Support)Venatorx Pharmaceuticals, Inc. (Grant/Research Support)Wockhardt Ltd. (Other Financial or Material Support, Research Collaborator) Robert A. Bonomo, MD, entasis (Research Grant or Support)Merck (Grant/Research Support)NIH (Grant/Research Support)VA Merit Award (Grant/Research Support)VenatoRx (Grant/Research Support)

Engineering the Prenyltransferase Domain of a Bifunctional Assembly-Line Terpene Synthase.

Copalyl diphosphate (CPP) synthase from Penicillium verruculosum (PvCPS) is a bifunctional diterpene synthase with both prenyltransferase and class II cyclase activities. The prenyltransferase α domain catalyzes the condensation of C5 dimethylallyl diphosphate with three successively added C5 isopentenyl diphosphates (IPPs) to form C20 geranylgeranyl diphosphate (GGPP), which then undergoes a class II cyclization reaction at the βγ domain interface to generate CPP. The prenyltransferase α domain mediates oligomerization to form a 648-kD (αβγ)6 hexamer. In the current study, we explore prenyltransferase structure-function relationships in this oligomeric assembly-line platform with the goal of generating alternative linear isoprenoid products. Specifically, we report steady-state enzyme kinetics, product analysis, and crystal structures of various site-specific variants of the prenyltransferase α domain. Crystal structures of the H786A, F760A, S723Y, S723F, and S723T variants have been determined at resolutions of 2.80, 3.10, 3.15, 2.65, and 2.00 Å, respectively. The substitution of S723 with bulky aromatic amino acids in the S723Y and S723F variants constricts the active site, thereby directing the formation of the shorter C15 isoprenoid, farnesyl diphosphate. While the S723T substitution only subtly alters enzyme kinetics and does not compromise GGPP biosynthesis, the crystal structure of this variant reveals a nonproductive binding mode for IPP that likely accounts for substrate inhibition at high concentrations. Finally, mutagenesis of the catalytic general acid in the class II cyclase domain, D313A, significantly compromises prenyltransferase activity. This result suggests molecular communication between the prenyltransferase and cyclase domains despite their distant connection by a flexible polypeptide linker.

Kinetic characterization and thermostability of C. elegans cytoplasmic and mitochondrial malate dehydrogenases

Malate dehydrogenase (MDH) catalyzes the conversion of NAD+ and malate to NADH and oxaloacetate in the citric acid cycle. Eukaryotes have one MDH isozyme that is imported into the mitochondria and one in the cytoplasm. We overexpressed and purified Caenorhabditis elegans cytoplasmic MDH-1 and mitochondrial MDH-2 in E. coli. Our goal was to compare the kinetic and structural properties of these enzymes because C. elegans can survive adverse environmental conditions, such as lack of food and elevated temperatures. In steady-state enzyme kinetics assays, we measured KM values for oxaloacetate of 54 and 52 μM and KM values for NADH of 61 and 107 μM for MDH-1 and MDH-2, respectively. We partially purified endogenous MDH-1 and MDH-2 from a mixed population of worms and separated them using anion exchange chromatography. Both endogenous enzymes had a KM for oxaloacetate similar to that of the corresponding recombinant enzyme. Recombinant MDH-1 and MDH-2 had maximum activity at 40 °C and 35 °C, respectively. In a thermotolerance assay, MDH-1 was much more thermostable than MDH-2. Protein homology modeling predicted that MDH-1 had more intersubunit salt-bridges than mammalian MDH1 enzymes, and these ionic interactions may contribute to its thermostability. In contrast, the MDH-2 homology model predicted fewer intersubunit ionic interactions compared to mammalian MDH2 enzymes. These results suggest that the increased stability of MDH-1 may facilitate its ability to remain active in adverse environmental conditions. In contrast, MDH-2 may use other strategies, such as protein binding partners, to function under similar conditions.

Steady-state enzyme kinetics

Steady-state enzyme kinetics is a cornerstone technique of biochemistry and related sciences since it allows the characterization and quantification of enzyme behaviour. Enzyme kinetics is widely used to investigate the physiological role of enzymes, determine the effects of mutations and characterize enzyme inhibitors. Well-known examples of enzyme inhibitors used to treat diseases include anti-infectives (e.g., penicillin, clavulanic acid and HIV protease inhibitors); anti-inflammatories (e.g., aspirin and ibuprofen); cholesterol-lowering statins; tyrosine kinase inhibitors used to treat cancer; and Viagra. Commonly, new disease treatments are discovered by using enzyme kinetics to identify the few active compounds residing within a large compound collection (‘high-throughput screening’). The subject of enzyme kinetics is typically introduced to first-year undergraduates with a mathematical description of behaviour. This Beginners Guide will give a brief overview of experimental enzyme kinetics and the characterization of enzyme inhibitors. Colorimetric assays using a microtitre plate will be considered, although most principles also apply to other assays.

Local interactions with the Glu166 base and the conformation of an active site loop play key roles in carbapenem hydrolysis by the KPC-2 β-lactamase

The Klebsiella pneumoniae carbapenemase-2 (KPC-2) is a common source of antibiotic resistance in Gram-negative bacterial infections. KPC-2 is a class A β-lactamase that exhibits a broad substrate profile and hydrolyzes most β-lactam antibiotics including carbapenems owing to rapid deacylation of the covalent acyl-enzyme intermediate. However, the features that allow KPC-2 to deacylate substrates more rapidly than non-carbapenemase enzymes are not clear. The active-site residues in KPC-2 are largely conserved in sequence and structure compared with non-carbapenemases, suggesting that subtle alterations may collectively facilitate hydrolysis of carbapenems. We utilized a nonbiased genetic approach to identify mutants deficient in carbapenem hydrolysis but competent for ampicillin hydrolysis. Subsequent pre–steady-state enzyme kinetics analyses showed that the substitutions slow the rate of deacylation of carbapenems. Structure determination via X-ray diffraction indicated that a F72Y mutant forms a hydrogen bond between the tyrosine hydroxyl group and Glu166, which may lower basicity and impair the activation of the catalytic water for deacylation, whereas several mutants impact the structure of the Q214-R220 active site loop. A T215P substitution lowers the deacylation rate and drastically alters the conformation of the loop, thereby disrupting interactions between the enzyme and the carbapenem acyl-enzyme intermediate. Thus, the environment of the Glu166 general base and the precise placement and conformational stability of the Q214-R220 loop are critical for efficient deacylation of carbapenems by the KPC-2 enzyme. Therefore, the design of carbapenem antibiotics that interact with Glu166 or alter the Q214-R220 loop conformation may disrupt enzyme function and overcome resistance.

Modeling human glucose-6-phosphate dehydrogenase mutations using C. elegans GSPD-1.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked, recessive condition that causes intermittent jaundice or hemolytic anemia because of low NADPH levels in red blood cells. We performed steady-state enzyme kinetics with the recombinant C. elegans ortholog of human G6PD, GSPD-1, and two mutants containing amino acid changes found in human patients. The KM values for glucose-6-phosphate were 100 ± 27 µM, 80 ± 22 µM, and 1000 ± 300 µM for the wild-type, D60N, and R252L GSPD-1 enzymes, respectively. The specific activities of the D60N and R252L mutants were 59% and 11%, respectively, of the wild-type value. Protein homology modeling suggested that the R252L mutation was more severe because the mutation caused a shift in the position of some active site residues. The D60N mutation may have affected the conformation of an outer loop of the enzyme. These data demonstrate that GSPD-1 is a promising model for human G6PD deficiencies, with the advantage that potential treatments could be studied in vivo in C. elegans.

Antagonism between substitutions in β-lactamase explains a path not taken in the evolution of bacterial drug resistance

CTX-M β-lactamases are widespread in Gram-negative bacterial pathogens and provide resistance to the cephalosporin cefotaxime but not to the related antibiotic ceftazidime. Nevertheless, variants have emerged that confer resistance to ceftazidime. Two natural mutations, causing P167S and D240G substitutions in the CTX-M enzyme, result in 10-fold increased hydrolysis of ceftazidime. Although the combination of these mutations would be predicted to increase ceftazidime hydrolysis further, the P167S/D240G combination has not been observed in a naturally occurring CTX-M variant. Here, using recombinantly expressed enzymes, minimum inhibitory concentration measurements, steady-state enzyme kinetics, and X-ray crystallography, we show that the P167S/D240G double mutant enzyme exhibits decreased ceftazidime hydrolysis, lower thermostability, and decreased protein expression levels compared with each of the single mutants, indicating negative epistasis. X-ray structures of mutant enzymes with covalently trapped ceftazidime suggested that a change of an active-site Ω-loop to an open conformation accommodates ceftazidime leading to enhanced catalysis. 10-μs molecular dynamics simulations further correlated Ω-loop opening with catalytic activity. We observed that the WT and P167S/D240G variant with acylated ceftazidime both favor a closed conformation not conducive for catalysis. In contrast, the single substitutions dramatically increased the probability of open conformations. We conclude that the antagonism is due to restricting the conformation of the Ω-loop. These results reveal the importance of conformational heterogeneity of active-site loops in controlling catalytic activity and directing evolutionary trajectories.

The effect of acetylation on isocitrate dehydrogenase 1 regulation

Isocitrate dehydrogenase 1 (IDH1) is a metabolic enzyme responsible for catalysis of isocitrate to α‐ketoglutarate using NADP+ as a cofactor. Cancerous mutations of IDH1 yield the neomorphic conversion of α‐ketoglutarate to D‐2‐hydroxyglutarate, a proposed oncometabolite. Various cancers such as gliomas, glioblastomas, acute myeloid leukemias, and bone cancers, are driven by IDH1 mutations. Although it has been established that IDH1 is an enzymatic driver of disease, more research is necessary to elucidate the molecular mechanisms of its regulation. Post‐translational modifications (PTMs), such as acetylation, serve as effective methods of protein regulation. Here, we hypothesize that acetylation may have a significant impact on IDH1 efficiency. To investigate this, we treated wild type IDH1 with acetyl group donors and generated wild type IDH1 mutants (K81Q, K224Q, K321Q) to mimic acetylation in a physiologically relevant setting. We then employed steady state enzyme kinetics to measure the rate of the normal reaction yielding α‐ketoglutarate. We report that acetylation has a notable effect on IDH1 catalytic efficiency in both the acetylation mimics and acetyl‐treated IDH1. Our data suggests that acetylation may be an essential post‐translational modification for regulating IDH1 catalytic efficiency, thus helping us understand pathways relevant to IDH1 activity.Support or Funding InformationThis work was funded by a Research Scholar Grant, RSG‐19‐075‐01‐TBE, from the American Cancer Society (C.D.S.), National Institutes of Health R00 CA187594 (C.D.S.), U54CA132384 (SDSU) & U54CA132379 (UC San Diego), MARC 5T34GM008303 (SDSU), and IMSD 5R25GM058906 (SDSU), as well as the California Metabolic Research Foundation (SDSU).

Off to a slow start: Analyzing lag phases and accelerating rates in steady-state enzyme kinetics

Steady-state enzyme kinetics typically relies on the measurement of ‘initial rates’, obtained when the substrate is not significantly consumed and the amount of product formed is negligible. Although initial rates are usually faster than those measured later in the reaction time-course, sometimes the speed of the reaction appears instead to increase with time, reaching a steady level only after an initial delay or ‘lag phase’. This behavior needs to be interpreted by the experimentalists. To assist interpretation, this article analyzes the many reasons why, during an enzyme assay, the observed rate can be slow in the beginning and then progressively accelerate. The possible causes range from trivial artifacts to instances in which deeper mechanistic or biophysical factors are at play. We provide practical examples for most of these causes, based firstly on experiments conducted with ornithine δ-aminotransferase and with other pyridoxal-phosphate dependent enzymes that have been studied in our laboratory. On the side to this survey, we provide evidence that the product of the ornithine δ-aminotransferase reaction, glutamate 5-semialdehyde, cyclizes spontaneously to pyrroline 5-carboxylate with a rate constant greater than 3 s−1.

Light-Regulation of Tryptophan Synthase by Combining Protein Design and Enzymology.

The spatiotemporal control of enzymes by light is of growing importance for industrial biocatalysis. Within this context, the photo-control of allosteric interactions in enzyme complexes, common to practically all metabolic pathways, is particularly relevant. A prominent example of a metabolic complex with a high application potential is tryptophan synthase from Salmonella typhimurium (TS), in which the constituting TrpA and TrpB subunits mutually stimulate each other via a sophisticated allosteric network. To control TS allostery with light, we incorporated the unnatural amino acid o-nitrobenzyl-O-tyrosine (ONBY) at seven strategic positions of TrpA and TrpB. Initial screening experiments showed that ONBY in position 58 of TrpA (aL58ONBY) inhibits TS activity most effectively. Upon UV irradiation, ONBY decages to tyrosine, largely restoring the capacity of TS. Biochemical characterization, extensive steady-state enzyme kinetics, and titration studies uncovered the impact of aL58ONBY on the activities of TrpA and TrpB and identified reaction conditions under which the influence of ONBY decaging on allostery reaches its full potential. By applying those optimal conditions, we succeeded to directly light-activate TS(aL58ONBY) by a factor of ~100. Our findings show that rational protein design with a photo-sensitive unnatural amino acid combined with extensive enzymology is a powerful tool to fine-tune allosteric light-activation of a central metabolic enzyme complex.

The heme-sensitive regulator SbnI has a bifunctional role in staphyloferrin B production by Staphylococcus aureus

Staphylococcus aureus infection relies on iron acquisition from its host. S. aureus takes up iron through heme uptake by the iron-responsive surface determinant (Isd) system and by the production of iron-scavenging siderophores. Staphyloferrin B (SB) is a siderophore produced by the 9-gene sbn gene cluster for SB biosynthesis and efflux. Recently, the ninth gene product, SbnI, was determined to be a free l-serine kinase that produces O-phospho-l-serine (OPS), a substrate for SB biosynthesis. Previous studies have also characterized SbnI as a DNA-binding regulatory protein that senses heme to control sbn gene expression for SB synthesis. Here, we present crystal structures at 1.9-2.1 Å resolution of a SbnI homolog from Staphylococcus pseudintermedius (SpSbnI) in both apo form and in complex with ADP, a product of the kinase reaction; the latter confirmed the active-site location. The structures revealed that SpSbnI forms a dimer through C-terminal domain swapping and a dimer of dimers through intermolecular disulfide formation. Heme binding had only a modest effect on SbnI enzymatic activity, suggesting that its two functions are independent and structurally distinct. We identified a heme-binding site and observed catalytic heme transfer between a heme-degrading protein of the Isd system, IsdI, and SbnI. These findings support the notion that SbnI has a bifunctional role contributing precursor OPS to SB synthesis and directly sensing heme to control expression of the sbn locus. We propose that heme transfer from IsdI to SbnI enables S. aureus to control iron source preference according to the sources available in the environment.

PS926 IN VITRO AND IN VIVO STUDIES OF NOVEL ASPARAGINASE MUTANTS FOR THE TREATMENT OF ACUTE LYMPHATIC LEUKEMIA

Background:E. coli derived L‐asparaginase (EcA) is an essential medicine in the combination chemotherapy for Acute Lymphoblastic Leukemia (ALL). Since it is exogenously derived, silent inactivation and hypersensitivity due to anti‐drug antibody have often necessitated discontinuation of the drug.Aims:Using a rational protein engineering approach, we created four EcA mutants with improved thermal stability, better serum stability, less antigenicity and less glutaminase activity. The mutant with most favourable pharmacological profile will be developed as a novel asparaginase for therapy of ALL.Methods:We identified and mutated several B‐cell epitopes and amino acid residues at the EcA interface that are responsible for activity, stability and antigenicity. Steady state enzyme kinetics was measured at 37oC (optimum temperature for EcA) and 62oC (EcA degrades at 65oC). The kinetic parameters, such as Km and Vmax, were calculated from linear fits of Michaelis‐Menten equation. Glutaminase activity of the mutants was measured and compared to the wild type EcA. The cytotoxicity of the EcA mutants was verified by performing MTT assay in ALL sensitive REH cell lines. Further indirect ELISA was performed to check the antigenicity of the mutants. The binding of the mutants to the commercially available l‐asparaginase antibody was analysed. Moreover, to evaluate the in vivo immunogenicity, mice were immunized with primary and booster doses of EcA mutants over 66 days followed by the measurement of IgG and IgM titers. In addition, the binding of wild‐type EcA and mutants to pre‐existing anti‐asparaginase antibodies present in ALL patient serum receiving asparaginase therapy was studied by indirect ELISA. Finally the activity‐time profile of the EcA mutants was plotted and was used to calculate the pharmacokinetic parameters, which were further compared with the commercially available l‐asparaginase drug.Results:At 37oC catalytic activity of the mutants was comparable to wild type. At 62oC, the catalytic activity of Mutant A, B and C was 155%, 96.5% and 98.2% respectively, compared to 56.9% for the wild type. All four mutants showed significantly lower glutaminase activity as compared to wild type EcA. In MTT assay, the wild type EcA showed 25.31% of cell kill, whereas mutant B showed highest cytotoxicity with 60% cell kill in Reh ALL cell lines. In terms of antigenicity, Mutant D was found to be 48% less antigenic compared to wild EcA, followed by Mutant B (26.4% less) and Mutant C (23.7% less). Pharmacokinetic profiling showed that all mutants had higher AUC and lower elimination rate constant, with Mutant B having highest AUC (1127.7 ± 363.6 vs. 72.12 ± 28.03 min∗ug/ml) and lowest elimination rate constant (0.048 ± 0.017 vs. 0.79 ± 0.42 ml/min) compared to commercially available L‐asparaginase. Mice immunized with Mutant B variant showed approximately 10‐fold lower IgG and IgM titers as compared to antibody responses induced by wild‐type EcA. Further, the binding of mutant B to serum antibody from the ALL patients was approximately 2‐fold less compared to wild‐type EcA.Summary/Conclusion:Considering all parameters, Mutant B was found to have the most favorable characteristics and emerged as the lead candidate for future development. This formulation is expected to overcome the existing deficiencies and clinical challenges encountered with the EcA formulations. Clinical development of this drug candidate is envisaged.

A Fold-Independent Interface Residue Is Crucial for Complex Formation and Allosteric Signaling in Class I Glutamine Amidotransferases.

The members of the glutamine amidotransferase (GATase) family catalyze the incorporation of ammonia within numerous metabolic pathways and can be categorized in two classes. Here, we concentrated on class I GATases, which are heteromeric enzyme complexes consisting of synthase subunits and glutaminase subunits with a catalytic Cys-His-Glu triad. Glutamine hydrolysis at the glutaminase subunit is (i) dependent on the formation of tight synthase-glutaminase complexes and (ii) allosterically coupled to the presence of the substrate at the synthase subunit. The structural basis of both complex formation and allostery is poorly understood. However, previous work on 4-amino-4-deoxychorismate synthase and imidazole glycerol phosphate synthase suggested that a conserved aspartate residue in their synthase subunits, which is located at the subunit interface close to the glutaminase catalytic triad, might be important for both features. We performed a computational screen of class I GATases from the Protein Data Bank and identified conserved and similarly located aspartate residues. We then generated alanine and glutamate mutants of these residues and characterized them by analytical gel filtration and steady-state enzyme kinetics. The results confirmed the important role of the wild-type aspartate residues for the formation of stable synthase-glutaminase complexes (in three of four cases) and the stimulation of glutaminase activity in the analyzed GATases (in all four cases). We present a model for rationalizing the dual role of the conserved aspartate residue toward a unifying regulation mechanism in the entire class I GATase family.

High-Throughput Virtual Screening, Molecular Dynamics Simulation, and Enzyme Kinetics Identified ZINC84525623 as a Potential Inhibitor of NDM-1.

The bacteria expressing New Delhi Metallo-β-lactamase-1 (NDM-1) can hydrolyze all β-lactam antibiotics including carbapenems, causing multi-drug resistance. The worldwide emergence and dissemination of gene blaNDM-1 (produces NDM-1) in hospital and community settings, rising problems for public health. Indeed, there is an urgent need for NDM-1 inhibitors to manage antibiotic resistance. Here, we have identified novel non-β-lactam ring-containing inhibitors of NDM-1 by applying a high-throughput virtual screening of lead-like subset of ZINC database. The screened compounds were followed for the molecular docking, the molecular dynamics simulation, and then enzyme kinetics assessment. The adopted screening procedure funnels out five novel inhibitors of NDM-1 including ZINC10936382, ZINC30479078, ZINC41493045, ZINC7424911, and ZINC84525623. The molecular mechanics-generalized born surface area and molecular dynamics (MD) simulation showed that ZINC84525623 formed the most stable complex with NDM-1. Furthermore, analyses of the binding pose after MD simulation revealed that ZINC84525623 formed two hydrogen bonds (electrostatic and hydrophobic interaction) with key amino acid residues of the NDM-1 active site. The docking binding free energy and docking binding constant for the ZINC84525623 and NDM-1 interaction were estimated to be −11.234 kcal/mol, and 1.74 × 108 M−1 respectively. Steady-state enzyme kinetics in the presence of ZINC84525623 show the decreased catalytic efficiency (i.e., kcat/Km) of NDM-1 on various antibiotics. The findings of this study would be helpful in identifying novel inhibitors against other β-lactamases from a pool of large databases. Furthermore, the identified inhibitor (ZINC84525623) could be developed as efficient drug candidates.