Determination Of Steady-state Kinetic Parameters Research Articles

Introducing Students to Biochemistry Through An Inquiry‐Based Curriculum Documented Using Electronic Notebooks on SciNote

In the laboratory section of the Biochemistry course at St. Mary's University, we perform a year‐long series of experiments; the first semester is a standard curriculum focused on teaching techniques and protein characteristics. The students learn to overexpress the enzyme tyrosine hydroxylase in bacteria, purify the enzyme, and after ten weeks, finish with determination of steady‐state kinetic parameters. The second semester is a CURE (curriculum‐based undergraduate research experience), facilitated by the students already knowing basic biochemistry techniques*. In the second semester we attempt to identify the functions of a handful of proteins that have been crystallized but whose activities are unknown. In order to simulate a real research lab, from the beginning, students are taught to keep notes using a research lab format rather than a teaching lab format. That is, emphasis is on data recording and analysis rather than on extensive background introductions. Further, to facilitate co‐operation and collaboration within and between student teams, we have integrated usage of the web electronic notebook program SciNote (scinote.net). SciNote is structured for a principal investigator of a tier one research team, but is flexible, so it is valuable to the teaching lab professor as well. SciNote allows table teams of students to share their data and ideas. After an exploration period with the features of SciNote, we have adopted a protocol in which students set up their own “projects”, which are filled with “experiments” that consist of “tasks”. “Tasks” are not what are usually considered “tasks”, rather, they are report sections such as materials, observations, data, results, discussion, and conclusion. The professor and all the students are enrolled in one large team, but smaller subteams, such as the ones that exist at each teaching lab table, can be set up. The professor particularly appreciates the simplicity and legibility of the reports that are automatically generated by the program after all text and data are entered. Further, the professor is able to monitor the progress and participation of each team member. It is hoped that once all the intricacies of SciNote usage are mastered, students at every campus will be able to compare findings and data on proteins that are being studied simultaneously across the country.Support or Funding Information* The inquiry curriculum is a shared project among several universities, and the delivery of the course differs in various regards at all of them. Those campuses and faculty colleagues include Rochester Institute of Technology (Drs. P. Craig, J. Mills, H. Bernstein, and S. O'Handley), Cal Poly San Luis Obispo (Drs. A. Goodman and A. McDonald), Ursinus College, (Dr. R. Roberts and N. Scharf) LaGrange College (Dr. A. Sikora), Purdue University (Dr. T Anderson and S. Irby), Hope College (Dr. M. Pikaart), and SUNY Oswego, (Drs. J. Koeppe and W. Kadima). The project is funded by NSF DUE‐1709592.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Determination of Steady-State Kinetic Parameters of 1-Deoxy-<I>D</I>-xylulose-5-phosphate Synthase by Pre-column Derivatization High Performance Liquid Chromatography Using 2,4 Dinitrophenylhydrazine as Derivative Reagent

利用含羰基化合物与2,4-二硝基苯肼在酸性条件下反应得到的产物腙对紫外-可见光有吸收的特性,采用柱前衍生高效液相色谱法测定了1-脱氧-D-木酮糖-5-磷酸合酶稳态动力学参数。酶反应产物1-脱氧-D-木酮糖-5-磷酸在碱性磷酸酶的作用下生成去磷酸化产物1-脱氧-D-木酮糖,然后与2,4-二硝基苯肼衍生成腙。衍生化反应的适宜条件为：HClO4浓度为1.5%,反应温度37℃,反应时间60 min,2,4-二硝基苯肼与1-脱氧-D-木酮糖-5-磷酸的摩尔比为6∶1。色谱条件：0～17 min,40%～80%甲醇;18～20 min,40%甲醇。结果表明,本方法具有较高的灵敏度和良好的线性关系,1-脱氧-D-木酮糖-5-磷酸的检出限为1.0 mg/L,在0.005～1.0 g/L范围内线性相关系数为0.999,相对标准偏差小于5.0%（n=3）,标准回收率为102.22%。用本方法测得的1-脱氧-D-木酮糖-5-磷酸合酶稳态动力学参数Km、Vmax和Kcat与文献报道一致。

Determination of Steady-State Kinetic Parameters of 1-Deoxy-D-xylulose-5-phosphate Synthase by Pre-column Derivatization High Performance Liquid Chromatography Using 2,4-Dinitrophenylhydrazine as Derivative Reagent

Abstract Carbonyl containing compounds can normally be determined by high performance liquid chromatography (HPLC) through pre-column derivatization with 2,4-dinitrophenylhydrazine (DNPH). Using this method, the steady-state kinetic parameters of 1-deoxy- D -xylulose-5-phosphate synthase (DXS) was measured. First, the enzymatic product 1-deoxy- D -xylulose-5-phosphate (DXP) was dephosphorylated by alkaline phosphatase, then the product 1-deoxy- D -xylulose (DX) was derived with DNPH in acidic solution to give the corresponding hydrazones which was subsequently determined by HPLC. The optimum derivatization conditions are as follows: 1.5% perchloric acid, reaction temperature of 37 °C, reaction time of 60min, molar ratio of DNPH to DXP 6:1. The HPLC was run with a linear gradient of methanol-water solvent system: 0 min, 40% methanol; 17 min, 80% methanol; 18 min, 40% methanol; 20 min, 40% methanol. The method has a detect limit of 1 mg L −1 for DXP and a linear correlation in the range of 0.005–1 g L −1 ( R = 0.999). The relative standard deviation is less than 5.0%. The steady-state kinetic parameters of DXS determined by this method are identical with the reported data.

A real‐time assay for monitoring Mycobacterium tuberculosis trehalose phosphate phosphatase activity

The otsB2 gene that encodes the enzyme trehalose phosphate phosphatase (TPP) is essential for the viability of Mycobacterium tuberculosis (M. tb). TPP possesses trehalose‐6‐phosphate (T6P) phosphatase activity that is central to the production of trehalose, which acts as the primary mycolate carrier molecule during biosynthesis of the mycobacterial outer membrane (MOM). The essentiality of the otsB2 gene and the role of TPP in trehalose biosynthesis makes TPP an intriguing target for antitubercular drug development. This abstract describes an enzymatic assay that allows real‐time monitoring of the TPP activity and determination of steady‐state kinetic parameters. The assay was analyzed for reproducibility and showed a Z′ value of 0.76 when using T6P at a concentration near the determined Km. The NIH clinical collection was used as a test set to determine assay consistency under typical assay conditions. The results from these experiments gave Z‐factors ranging from 0.44–0.52. Taken together, these data show the assay to be rapid, highly reproducible, and amenable to high‐throughput screening methodologies as a basis for identifying lead compounds that inhibit TPP.

Distribution and Biochemical Properties of an M1-family Aminopeptidase in Plasmodium falciparum Indicate a Role in Vacuolar Hemoglobin Catabolism

Aminopeptidases catalyze N-terminal peptide bond hydrolysis and occupy many diverse roles across all domains of life. Here we present evidence that an M1-family aminopeptidase, PfA-M1, has been recruited to specialized roles in the human malaria parasite Plasmodium falciparum. PfA-M1 is abundant in two subcellular compartments in asexual intraerythrocytic parasites; that is, the food vacuole, where the catabolism of host hemoglobin takes place, and the nucleus. A unique N-terminal extension contributes to the observed dual targeting by providing a signal peptide and putative alternate translation initiation sites. PfA-M1 exists as two major isoforms, a nuclear 120-kDa species and a processed species consisting of a complex of 68- and 35-kDa fragments. PfA-M1 is both stable and active at the acidic pH of the food vacuole lumen. Determination of steady-state kinetic parameters for both aminoacyl-β-naphthylamide and unmodified dipeptide substrates over the pH range 5.0-8.5 reveals that k(cat) is relatively insensitive to pH, whereas K(m) increases at pH values below 6.5. At the pH of the food vacuole lumen (5.0-5.5), the catalytic efficiency of PfA-M1 remains high. Consistent with the kinetic data, the affinity of peptidic competitive inhibitors is diminished at acidic pH. Together, these results support a catalytic role for PfA-M1 in the food vacuole and indicate the importance of evaluating the potency of peptidic inhibitors at physiologically relevant pH values. They also suggest a second, distinct function for this enzyme in the parasite nucleus.

Acyl substrate preferences of an IAA-amido synthetase account for variations in grape (Vitis vinifera L.) berry ripening caused by different auxinic compounds indicating the importance of auxin conjugation in plant development

Nine Gretchen Hagen (GH3) genes were identified in grapevine (Vitis vinifera L.) and six of these were predicted on the basis of protein sequence similarity to act as indole-3-acetic acid (IAA)-amido synthetases. The activity of these enzymes is thought to be important in controlling free IAA levels and one auxin-inducible grapevine GH3 protein, GH3-1, has previously been implicated in the berry ripening process. Ex planta assays showed that the expression of only one other GH3 gene, GH3-2, increased following the treatment of grape berries with auxinic compounds. One of these was the naturally occurring IAA and the other two were synthetic, α-naphthalene acetic acid (NAA) and benzothiazole-2-oxyacetic acid (BTOA). The determination of steady-state kinetic parameters for the recombinant GH3-1 and GH3-2 proteins revealed that both enzymes efficiently conjugated aspartic acid (Asp) to IAA and less well to NAA, while BTOA was a poor substrate. GH3-2 gene expression was induced by IAA treatment of pre-ripening berries with an associated increase in levels of IAA-Asp and a decrease in free IAA levels. This indicates that GH3-2 responded to excess auxin to maintain low levels of free IAA. Grape berry ripening was not affected by IAA application prior to veraison (ripening onset) but was considerably delayed by NAA and even more so by BTOA. The differential effects of the three auxinic compounds on berry ripening can therefore be explained by the induction and acyl substrate specificity of GH3-2. These results further indicate an important role for GH3 proteins in controlling auxin-related plant developmental processes.

Comparison between Two Erwinia carotovora L-Asparaginase II Constructions: cloning, Heterologous Expression, Purification, and Kinetic Characterization

L-Asparaginase II from Erwinia carotovora may represent an important alternative therapy in the treatment of acute childhood lymphoblastic leukaemia, despite its promising lower glutaminase activity than Escherichia coli and Erwinia chrysanthemi L-asparaginases II, currently used in treatment of this disease. Here we describe cloning, expression, purification and determination of steady-state kinetic parameters for E. carotovora L-asparaginase II: with (AspSP) and without the signal peptide (AspMP). AspMP was purified to homogeneity by a single-step protocol with 91% yield, and AspSP by a two-step protocol with 28% yield. In addition, both enzymes presented similar high specific activities: 208.1 and 237.6 U mg-1, respectively. The Km and kcat values showed that AspMP has lower glutaminase activity than AspSP. Moreover AspMP is produced by a simpler purification protocol, and at higher yield. This process can be amenable to large scale production and be of interest to researchers and biopharmaceutical companies

A continuous microplate assay for sirtuins and nicotinamide-producing enzymes

Nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylases (sirtuins) and other enzymes that produce nicotinamide are integral to many cellular processes. Yet current activity measurements involve expensive and time-consuming assays. Here we present a spectroscopic assay that circumvents many issues of previous methods. This assay permits continuous product monitoring over time, allows determination of steady-state kinetic parameters, and is readily adaptable to high-throughput screening. The methodology uses an enzyme-coupled system in which nicotinamide is converted to nicotinic acid and ammonia by nicotinamidase. The ammonia is transferred to α-ketoglutarate via glutamate dehydrogenase, yielding glutamate and the oxidation of NAD(P)H to NAD(P)+, which is measured spectrophotometrically at 340nm. Using this continuous assay with sirtuin-1 (Sirt1) and the ADP-ribosyl cyclase CD38, the resulting steady-state kinetic parameters are in excellent agreement with values obtained by other published methods. Importantly, this assay permitted determination of kcat and Km values with the native acetylated substrate acetyl-CoA synthetase-1; measurement of Sirt1, Sirt2, and Sirt3 activities from mammalian cell extracts; and determination of IC50 values of various Sirt1 inhibitors. This assay is applicable to any nicotinamide-forming enzyme and will be an important tool to address many outstanding questions surrounding their regulation.

Tuning the Strength of a Bacterial N-end Rule Degradation Signal

The N-end rule is a degradation pathway conserved from bacteria to mammals that links a protein's stability in vivo to the identity of its N-terminal residue. In Escherichia coli, the components of this pathway directly responsible for protein degradation are the ClpAP protease and its adaptor ClpS. We recently demonstrated that ClpAP is able to recognize N-end motifs in the absence of ClpS although with significantly reduced substrate affinity. In this study, a systematic sequence analysis reveals new features of N-end rule degradation signals. To achieve specificity, recognition of an N-end motif by the protease-adaptor complex uses both the identity of the N-terminal residue and a free alpha-amino group. Acidic residues near the first residue decrease substrate affinity, demonstrating that the identity of adjacent residues can affect recognition although significant flexibility is tolerated. However, shortening the distance between the N-end residue and the stably folded portion of a protein prevents degradation entirely, indicating that an N-end signal alone is not always sufficient for degradation. Together, these data define in vitro the sequence and structural requirements for the function of bacterial N-end signals.

Determination of steady-state kinetic parameters for a xylanase-catalyzed hydrolysis of neutral underivatized xylooligosaccharides by mass spectrometry

A direct mass spectrometric approach was used for the determination of steady-state kinetic parameters, the turnover number ( k cat), the Michaelis constant ( K M), and the specificity constant ( k cat/ K M) for an enzyme-catalyzed hydrolysis of xylooligosaccharides. Electrospray ionization mass spectrometry was performed to observe product distributions and to determine k cat, K M, and k cat/ K M values for Trichoderma reesei endo-1,4-β-xylanase II (TRX II) with xylohexaose (Xyl 6), xylopentaose (Xyl 5), xylotetraose (Xyl 4), and xylotriose (Xyl 3) as substrates. The determined k cat/ K M values (0.93, 0.37, 0.027, and 0.00015 μM −1 s −1, respectively) indicated that Xyl 6 was the most preferred substrate of TRX II. In addition, the obtained K M value for Xyl 5 (136 μM) was roughly twice as high as that for Xyl 6 (73 μM), suggesting that at least six putative subsites contribute to the substrate binding in the active site of TRX II. Previous mass spectrometric assays for enzyme kinetics have been used mostly in the case of reactions that result in a transfer of acidic groups (e.g., phosphate) into neutral oligosaccharides giving rise to negatively charged products. Here we demonstrate that such analysis is also feasible in the case of neutral underivatized oligosaccharides. Implications of the results for the catalytic mechanism of TRX II in particular are discussed.

The role for an invariant aspartic acid in hypoxanthine phosphoribosyltransferases is examined using saturation mutagenesis, functional analysis, and X-ray crystallography.

The role of an invariant aspartic acid (Asp137) in hypoxanthine phosphoribosyltransferases (HPRTs) was examined by site-directed and saturation mutagenesis, functional analysis, and X-ray crystallography using the HPRT from Trypanosoma cruzi. Alanine substitution (D137A) resulted in a 30-fold decrease of k(cat), suggesting that Asp137 participates in catalysis. Saturation mutagenesis was used to generate a library of mutant HPRTs with random substitutions at position 137, and active enzymes were identified by complementation of a bacterial purine auxotroph. Functional analyses of the mutants, including determination of steady-state kinetic parameters and pH-rate dependence, indicate that glutamic acid or glutamine can replace the wild-type aspartate. However, the catalytic efficiency and pH-rate profile for the structural isosteric mutant, D137N, were similar to the D137A mutant. Crystal structures of four of the mutant enzymes were determined in ternary complex with substrate ligands. Structures of the D137E and D137Q mutants reveal potential hydrogen bonds, utilizing several bound water molecules in addition to protein atoms, that position these side chains within hydrogen bond distance of the bound purine analogue, similar in position to the aspartate in the wild-type structure. The crystal structure of the D137N mutant demonstrates that the Asn137 side chain does not form interactions with the purine substrate but instead forms novel interactions that cause the side chain to adopt a nonfunctional rotamer. The results from these structural and functional analyses demonstrate that HPRTs do not require a general base at position 137 for catalysis. Instead, hydrogen bonding sufficiently stabilizes the developing partial positive charge at the N7-atom of the purine substrate in the transition-state to promote catalysis.

Kinetics of hydrolysis of dansyl peptide substrates by thermolysin: analysis of fluorescence changes and determination of steady-state kinetic parameters.

The stopped-flow fluorescence technique has been used to study the hydrolysis of 10 dansyl peptides by thermolysin. The origin of the fluorescence changes observed during the reactions has been investigated in detail. Depending on the substrate and the excitation wavelength, the dansyl fluorescence changes observed arise either from energy transfer (maximal at lambda ex = 230 and 280 nm) between Trp residues of thermolysin and the dansyl group of the substrate in enzyme-substrate (ES) complexes or from both sources. These excitation (maximal at lambda ex = 245 and 340 nm) of the free substrate and product, or from both sources. These two types of fluorescence signals reflect the concentrations of ESi and free substrate, respectively. Both types of fluorescence changes have been used to monitor the reaction progress, and different mathematical formalisms have been used to determine the kinetic parameters for the reactions with results that are in good agreement. The efficiency of Trp quenching by a series of five dansyl tripeptides is shown to be related to the fractional saturation of enzyme and follows the KM-1 values for the substrates. The quenching efficiency for a dansyl tetrapeptide is weaker due to the greater distance between the dansyl group and the Trp-115 donor in thermolysin. On the basis of these studies, substrates capable of supporting more detailed kinetic studies of thermolysin have been identified.

Mechanistic studies of peptidyl prolyl cis-trans isomerase: evidence for catalysis by distortion

Cyclophilin, the cytosolic binding protein for the immunosuppressive drug cyclosporin A, has recently been shown to be identical with peptidyl prolyl cis-trans isomerase [Fischer, G., Wittmann-Liebold, B., Lang, K., Kiefhaber, T., & Schmid, F.X. (1989) Nature 337, 476; Takahashi, N., Hayano, T., & Suzuki, M. (1989) Nature 337, 473]. To provide a mechanistic framework for studies of the interaction of cyclophilin with cyclosporin, we investigated the mechanism of the PPI-catalyzed cis to trans isomerization of Suc-Ala-Xaa-cis-Pro-Phe-pNA (Xaa = Ala, Gly). Our mechanistic studies of peptidyl prolyl cis-trans isomerase include the determination of steady-state kinetic parameters, pH and temperature dependencies, and solvent and secondary deuterium isotope effects. The results of these experiments support a mechanism involving catalysis by distortion in which the enzyme uses free energy released from favorable, noncovalent interactions with the substrate to stabilize a transition state that is characterized by partial rotation about the C-N amide bond.

A direct spectrophotometric assay for d-alanine carboxypeptidases and for the esterase activity of β-lactamases

A direct spectrophotometric pH indicator method has been devised to assay the activity of the d-Ala carboxypeptidase/transpeptidase of Streptomyces R61, and which should be of general application to d-Ala carboxypeptidases. The substrate employed is N,N′-diacetyl- l-lysyl- d-alanyl- d-lactate. The method allows the determination of steady-state kinetic parameters, and can also be used for the assay of the esterase activity of β-lactamases against specific depsipeptides.