The reliability of Michaelis constants and maximum velocities estimated by using the integrated Michaelis–Menten equation

1. Experimental progress curves were simulated for a reaction obeying Michaelis-Menten kinetics. 2. K(m) and V were estimated (a) by fitting the integrated Michaelis-Menten equation to the progress curves, and (b) from the initial slopes of the curves (i.e. from initial velocities). 3. The integrated equation could not be fitted successfully by a non-linear method, so it was transformed and fitted by a linear method. 4. Provided that the initial substrate concentration was greater than K(m) and the data were precise enough, the integrated equation gave parameter estimates which were unbiased and as reliable as those derived from initial velocities although based on fewer experiments. 5. The integrated equation could be used for progress curves of unknown origin.     

Antithrombin III assay using a chromogenic substrate in newborns

The assay of antithrombin III activity (AT-III) was performed by chromogenic synthetic substrate in 42 full-term newborns, 1 to 7 days aged. AT-III levels were lower than normal adults in the first day of life and lessened again in the 3rd day. After this period the levels of AT-III rose slowly and in the 7th day were about half the concentration of adults.     

A chromogenic substrate for a beta-xylosidase-coupled assay of alpha-glucuronidase

4-Nitrophenyl 2-(4-O-methyl-alpha-d-glucopyranuronosyl)-beta-d-xylopyranoside obtained on deesterification of 4-nitrophenyl 2-O-(methyl 4-O-methyl-alpha-d-glucopyranosyluronate)-beta-d-xylopyranoside (Hirsch et al., Carbohydr. Res. 310, 145-149, 1998) was found to be an excellent substrate for the measurement of hemicellulolytic alpha-glucuronidase activity. A new precise alpha-glucuronidase assay was developed by coupling the alpha-glucuronidase-catalyzed formation of 4-nitrophenyl beta-d-xylopyranoside with its efficient hydrolysis by beta-xylosidase. A recombinant strain of Saccharomyces cerevisiae, harboring and expressing the beta-xylosidase gene xlnD of Aspergillus niger under control of the alcohol dehydrogenase II promoter on a multicopy plasmid, was used as a source of beta-xylosidase. The activity values of beta-xylosidase in the assay required to achieve a steady-state rate of 4-nitrophenol formation shortly after starting the alpha-glucuronidase reaction were obtained both experimentally and by calculation using the kinetics of coupled enzyme reactions.     

Assay of the high-alkaline proteinase from alkalophilic bacillus PB92 using a chromogenic tripeptide substrate

The chromogenic tripeptide substrate, benzyloxycarbonylglycyl-L-prolyl-L-citrulline p-nitroanilide, is proposed for the assay of the high-alkaline proteinase, HAP-PB92, from an alkalophilic Bacillus. The assay method is sensitive, reproducible, and may be adapted for an automatic system.     

The muscle pattern of a segment of Drosophila may be determined by neurons and not by contributing myoblasts

Each segment of Drosophila has a characteristic pattern of muscles. Like the segments of the cuticle and the central nervous system, the muscle pattern is ultimately dependent on the deployment of selector genes such as elements of the bithorax complex. We use nuclear transplantation to make genetic mosaics in which the donor, but not the host, is mutant for part of the bithorax complex. Making use of a muscle pattern that is found only in the male, we ask which cells have to be mutant in order to obtain mutant muscles and find that these crucial cells do not contribute to the muscles themselves. The evidence implicates neurons that innervate the muscles. Our hypothesis is that the sex and segmental identity of the motor or neurosecretory neurons determine the development of muscle pattern.     

Deep learning allows genome-scale prediction of Michaelis constants from structural features

The Michaelis constant K_M describes the affinity of an enzyme for a specific substrate and is a central parameter in studies of enzyme kinetics and cellular physiology. As measurements of K_M are often difficult and time-consuming, experimental estimates exist for only a minority of enzyme–substrate combinations even in model organisms. Here, we build and train an organism-independent model that successfully predicts K_M values for natural enzyme–substrate combinations using machine and deep learning methods. Predictions are based on a task-specific molecular fingerprint of the substrate, generated using a graph neural network, and on a deep numerical representation of the enzyme’s amino acid sequence. We provide genome-scale K_M predictions for 47 model organisms, which can be used to approximately relate metabolite concentrations to cellular physiology and to aid in the parameterization of kinetic models of cellular metabolism.

To understand the action of an enzyme, we need to know its affinity for its substrates, quantified by Michaelis constants, but these are difficult to measure experimentally. This study shows that a deep learning model that can predict them from structural features of the enzyme and substrate, providing KM predictions for all enzymes across 47 model organisms.     

Determination of kinetic constants in the general case of cooperative type or Michaelis enzymes inhibited by their substrate: theoretic analysis

For an enzyme (E) susceptible to substrate (S) inhibition, (S) can bind on one hand to (E) and on the other hand to (ES), leading to the dead-end complexes (SE) and (SES). In the general case where the (E)/(S) interaction obeys the Hill equation, the theoretical maximum velocity VM can be estimated when n not equal to 1, from the determination of velocities v beta at substrate concentrations S beta = Sm beta where Sm is the value corresponding to the actual maximum velocity vm. The Hill coefficient (n) as well as the constants KS, KSE and KSES corresponding to the respective dissociations of the complexes (ES), (SE) and (SES) are then determined from the equation: Ln (v/(VM-v] = nLnS-LnKS(1 + Sn/KSE + S2n/KS KSES) and its two asymptotes.     

Preferred step frequency during downhill running may be determined by muscle activity

It is well established that metabolic cost is minimized at an individual’s running preferred step frequency (PSF). It has been proposed that the metabolic minimum at PSF is due to a tradeoff between mechanical factors, however, this ignores muscle activity, the primary consumer of energy. Thus, we hypothesized that during downhill running, total muscle activity would be greater with deviations from PSF. Specifically, we predicted that slow step frequencies would have greater stance activity while fast step frequencies would have greater swing activity. We collected metabolic cost and leg muscle activity data while 10 healthy young adults ran at 3.0 m/s for 5 min at level and downhill at PSF and ±15% PSF. In support of our hypothesis, there was a significant main effect for step frequency for both metabolic cost and total muscle activity. In addition, there was greater muscle activity in the stance phase during the slower step frequency while muscle activity was greater in the swing phase during the fast step frequency. This suggests that PSF is partially determined by the tradeoff between the greater cost of muscle activity in the swing phase and lower cost in the stance phase with faster step frequency.     

Development of a dual fluorogenic and chromogenic dipeptidyl peptidase IV substrate

A new far-red dual fluorogenic and chromogenic substrate, 5-glycylprolylglycylprolyl-9-di-3-sulfonyl-propylaminobenza[a]phenoxazonium perchlorate (GPGP-2SBPO), was developed for dipeptidyl peptidase IV (DPP-IV) sensing. The glycylprolylglycylprolyl tetrapeptide was chosen as the recognition sequence due to its stability under physiological conditions. In contrast, the truncated substrate, GP-2SBPO, containing only a glycylprolyl peptide, is unstable. Proteolysis of GPGP-2SBPO was assayed by monitoring the absorbance and fluorescence signals from the released fluorochrome, 2SBPO, at 625 and 670nm, respectively.     

The global average DNA base composition of coding regions may be determined by the electron-ion interaction potential

It is shown how one can understand the frequencies of occurrence of nucleotides in coding DNA sequences on the basis of electron-ion interaction potential.     

Kinetic properties of bovine brain proteinl-isoaspartyl methyltransferase determined using a synthetic isoaspartyl peptide substrate

Proteinl-isoaspartyl methyltransferase, an enzyme enriched in brain, is implicated in the repair of age-damaged proteins containing atypical, isoaspartyl peptide bonds. We have investigated the kinetics of methylation using a synthetic peptide substrate having the structure Trp-Ala-Gly-Gly-isoAsp-Ala-Ser-Gly-Glu. Double-reciprocal plots of initial velocity versus concentration of S-adenosylmethionine (AdoMet) at different fixed concentrations of peptide gave straight lines converging at a positive 1/v value and a negative 1/AdoMet value. The product S-adenosylhomocysteine (AdoHcy) was a competitive inhibitor towards AdoMet and a linear mixed-type inhibitor towards peptide. These results are consistent with the rapid-equilibrium random sequential bi-bi mechanism previously proposed for the enzyme, but they also reveal the formation of the deadend, enzyme-peptide-AdoHcy, complex. The rate constants were:V _max=32–34 nmol/min/mg,K ^peptide=7.6–9.4 M,K ^AdoMet=1.9–2.2 M, =0.43–0.53,K ^AdoHcy=0.08 M, =2.9. The interaction factors and indicate that binding of enzyme to peptide increases its affinity for AdoMet and decreases its affinity for AdoHcy. Methylation was linear with time throughout the transfer of 2 mol of methyl groups/mol of enzyme. This absence of burst kinetics suggests that slow release of products cannot explain the low turnover number.Special issue dedicated to Dr. Paul Greengard     

The approach to the Michaelis complex in lactate dehydrogenase: the substrate binding pathway

We examine here the dynamics of forming the Michaelis complex of the enzyme lactate dehydrogenase by characterizing the binding kinetics and thermodynamics of oxamate (a substrate mimic) to the binary lactate dehydrogenase/NADH complex over multiple timescales, from nanoseconds to tens of milliseconds. To access such a wide time range, we employ standard stopped-flow kinetic approaches (slower than 1 ms) and laser-induced temperature-jump relaxation spectroscopy (10 ns-10 ms). The emission from the nicotinamide ring of NADH is used as a marker of structural transformations. The results are well explained by a kinetic model that has binding taking place via a sequence of steps: the formation of an encounter complex in a bimolecular step followed by two unimolecular transformations on the microsecond/millisecond timescales. All steps are well described by single exponential kinetics. It appears that the various key components of the catalytically competent architecture are brought together as separate events, with the formation of strong hydrogen bonding between active site His(195) and substrate early in binding and the closure of the catalytically necessary protein surface loop over the bound substrate as the final event of the binding process. This loop remains closed during the entire period that chemistry takes place for native substrates; however, motions of other key molecular groups bringing the complex in and out of catalytic competence appear to occur on faster timescales. The on-enzyme K(d) values (the ratios of the microscopic rate constants for each unimolecular step) are not far from one. Either substantial, approximately 10-15%, transient melting of the protein or rearrangements of hydrogen bonding and solvent interactions of a number of water molecules or both appear to take place to permit substrate access to the protein binding site. The nature of activating the various steps in the binding process seems to be one overall involving substantial entropic changes.     

Study on a new chromogenic substrate for the prothrombin time determination

The aim of our study was to evaluate the possibility of using a chromogenic substrate for the prothrombin time determination. The reagent used by us (Chromoquick) is composed of a human placenta thromboplastin and chromogenic substrate (Tos-Gly-Pro-Arg-5-amino-2-nitrobenzoic acid-isopropylamide), calcium chloride and a buffer. Normal subjects, patients with liver disease, patients on oral anticoagulant therapy, patients on heparin therapy, heterozygous and homozygous patients for prothrombin complex defects and other miscellaneous conditions have been investigated. The results of chromoquick have been related with standard prothrombin time obtained using a human placenta thromboplastin (Thromborel) and rabbit brain and lung thromboplastin (Simplastin). The normal range was 18-23 s for chromoquick and 13.5-15.5 s for the standard prothrombin times using Thromborel and Simplastin. In all groups of patients examined we noticed a significant correlation between the chromogenic and the classic prothrombin times with r values varying between +0.505 and +0.947. The statistical significance resulted from p values varying between less than 0.05 and less than 0.001. Only in the case of some heterozygotes for prothrombin complex factor defects the values obtained have not been unequivocal in the sense that in a few instances the heterozygotes seemed to escape detection. Therefore, it seems that the introduction of chromogenic substrates in laboratory practice for the prothrombin time determination is possible and can offer considerable advantages like standardization and automation. The only disadvantage may be caused by costs involved.     

Kinetic constants of signal peptidase I using cytochrome b5 as a precursor substrate.

A procedure is described for measuring Escherichia coli signal peptidase I activity which exploits an intact precursor protein composed of the alkaline phosphatase signal peptide fused to the full length mammalian cytochrome b5. This cytochrome b5 precursor protein has been extensively characterised and shown to be processed accurately by purified signal peptidase I [Protein Expr. Purif. 7 (1996) 237]. The amphipathic, chimaeric cytochrome b5 precursor was isolated in mg quantities in a highly homogeneous state under non-denaturing conditions. The processing of the cytochrome b5 precursor by signal peptidase displayed Michaelis-Menten kinetics with K(m)=50 microM and k(cat)=11 s(-1). The K(m) was 20-fold lower than that obtained with signal peptide substrates and 3-fold higher than that reported for pro-OmpA-nuclease A precursor fusion. The corresponding turnover number, k(cat), was four orders of magnitude greater than the peptide substrates but was 2-fold lower than pro-OmpA-nuclease A precursor fusion. These results confirm that both the affinities and the catalytic power of the signal peptidase are significantly higher for macromolecular precursor substrates than for the shorter signal peptide substrates.     

Calculating affinity constants of substrate mixtures in a chemostat

An “oversimplified” method for calculating affinity constants in substrate mixtures is evaluated concerning its validity. It is shown that the real affinity to the mixture as determined by using the correct method is always higher, i.e., leads to a lower affinity constant, as compared to the wrong method frequently used.     

The structure of a Michaelis serpin-protease complex.

Serine protease inhibitors (serpins) regulate the activities of circulating proteases. Serpins inhibit proteases by acylating the serine hydroxyl at their active sites. Before deacylation and complete proteolysis of the serpin can occur, massive conformational changes are triggered in the serpin while maintaining the covalent linkage between the protease and serpin. Here we report the structure of a serpin-trypsin Michaelis complex, which we visualized by using the S195A trypsin mutant to prevent covalent complex formation. This encounter complex reveals a more extensive interaction surface than that present in small inhibitor-protease complexes and is a template for modeling other serpin-protease pairs. Mutations of several serpin residues at the interface reduced the inhibitory activity of the serpin. The serine residue C-terminal to the scissile peptide bond is found in a closer than usual interaction with His 57 at the active site of trypsin.     

Histochemical demonstration of mouse submandibular esterproteases with a new chromogenic substrate.

A new method for the histochemical demonstration of mouse submandibular esterproteases has been developed. The substrate is N-acetyl-L-methionine alpha-naphthyl ester. The main enzyme reaction was found in the apical region of the secretory tubules with a marked sex difference as expected. First attempts were made to differentiate histochemically between the various esterproteolytic enzymes.