Kinetic properties of derepressible acid phosphatase from the yeast from of Yarrowia lipolytica

(1) An acid phosphatase from depressed cells of the yeast form of Yarrowia lipolytica has been characterized kinetically by studies on specificity, inhibition, rate equation forms and modelling of the enzyme mechanisms. (2) The study on specificity revealed that the acid phosphatase is a rather unspecific phosphohydrolase that has similar activity on several different phosphate esters. A very weak transphosphorylating activity was also detected. (3) Among the reversible inhibitors, phosphate and vanadate were outstanding, whereas EDTA behaved as an activator. (4) v vs. [S] studies with o-carboxyphenyl phosphate as substrate show that the acid phosphatase of Y. lipolytica exhibits non-Michaelian behaviour, a minimum degree of 2:2 being detected for the rate equation in [S]. (5) The inhibition by phosphate and vanadate seems to have the same pattern of partial inhibition with a certain non-competitive nature, 1:1 being the minimum degree of the rate equation detected in (I).     

Bovine lens aldehyde reductase (aldose reductase). Purification, kinetics and mechanism.

Aldehyde reductase (aldose reductase) was purified to homogeneity (as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis) from bovine lens by affinity chromatography on NADP+-Sepharose. The enzyme, a monomer of Mr about 40000, was active with a variety of alpha- hydroxyketones , including fructose. The minimum degree of the rate equation was 2:2 in the case of DL-glyceraldehyde, but linear kinetics were observed for glucose and NADPH over the concentration range studied. The enzyme largely followed a ternary-complex mechanism, with initial binding of NADPH before glucose and final release of NADP+.     

A microcomputer method for designing optimal experiments for estimating enzyme kinetic parameters

A computer program, written in BASIC, for designing optimal experiments with the aim of evaluating estimates of the parameters for any enzyme kinetic model is given. This computer program can be run on any microcomputer with less than 32 Kbytes of random access memory. The program uses the termed D-optimization design criterion, which minimizes the determinant of the variance-covariance matrix. The user only supplies the rate equation, the maximum and minimum concentrations of substrates and inhibitors, the weighting pattern, and the best possible values of the parameters. The computer supplies the optimal substrate and inhibitor concentrations (one for each parameters), for estimating the parameter values, and the determinant of the variance-covariance matrix. Likewise, the microcomputer supplies the eigenvalues and eigenvectors of information and redundancy matrices, the sensitivity and the global redundancy.     

Protein kinase catalytic subunit (PKAcat) from bovine lens: purification, characterization and phosphorylation of lens crystallins

The purification and functional characterization of protein kinase A catalytic subunit (PKAcat) from bovine lens cytosol has been described. Purification to homogeneity has been achieved by using 100 kDa cut-off membrane filtration followed by Sephacryl S-300 chromatography and finally fractionating on High Q anion exchange column. The purified protein migrates as a single band of molecular mass ∼41 kDa on 12.5% SDS-PAGE. Proteomic data from ion trap LC-MS when analyzed through NCBI blast program reveals significant homology (52%) with bovine zeta-crystallin and also some homology with pig casein kinase I alpha chain (38%) and SLA-DR1 beta 1 domain (38%). The search does not indicate homology with any known catalytic subunit of PKA. Inspite of the significant homology with the zeta-crystallin, our protein is different from it in terms of molecular mass. pI value of the kinase (5.3) obtained from 2D analysis is also different from zeta-crystallin (8.5). The protein is found to contain 17% α-helix, 26.5% β-sheet, 21.4% turn and 34.7% random coil. The active catalytic subunit of the bovine lens cAMP-dependent kinase belongs to Type I Cα subtype. The enzyme shows maximum activity at 30 min incubation in presence of 5 mM MgCl₂ and 50 μM ATP. The kinase shows broad substrate specificity. It prefers Ser over Thr as phosphorylating residue. Phosphorylation of crystallin proteins, major protein fraction of bovine lens and phosphorylation of chaperone protein α crystallin by the kinase suggests that the kinase plays some crucial role in regulation of chaperone function within lens.     

Evidence concerning a possible steady state rate equation for E. coli alkaline phosphatase

• 1.1. Steady state data was obtained for alkaline phosphatase over a wide range of experimental conditions using two substrates, four inhibitors, two modifiers and several pH, ionic strength and temperatures values.
• 2.2. The data was fitted by rational functions of degree 1:1, 2:2 and 3:3 using a non-linear regression program and then the F-test was used to assess the goodness of fit.
• 3.3. A proportion of the curves could only be fitted by 2:2 functions but many of them could be adequately fitted by 1:1 functions.
• 4.4. No statistically significant improvement in fit occurred with 3:3 functions.
• 5.5. Data was simulated using a computer program to see what sort of curves could be generated by a two sites mechanism proposed for alkaline phosphatase and this study showed that it is difficult to detect cubic terms in this rate equation.
• 6.6. It was concluded that alkaline phosphatase does not obey Michaelis-Menten kinetics. Rather, the steady state data require a mechanism of at least second degree but do not exclude a rate equation of third degree.

     

Kinetics of action of chymosin (rennin) on some peptide bonds of bovine beta-casein

The first steps of proteolysis of bovine beta-casein by chymosin were studied quantitatively by using reverse-phase high-performance liquid chromatography (RP-HPLC). Although chymosin has a broad specificity, it has been possible to selectively study the hydrolysis of two bonds (Ala-189-Phe-190 and Leu-192-Tyr-193) by choosing appropriate conditions. The disappearance of the substrate and the appearance of the reaction products as a function of time were followed at 220 nm by RP-HPLC. For concentrations where beta-casein was in a micellar form, the Michaelian parameters corresponding to the cleavage of bond 192-193 were determined by measuring initial rates of reaction at different substrate concentrations in a time period for which splitting of bond 189-190 was negligible. The following results were obtained; k1cat = 1.54 s-1, K1m = 0.075 mM, and k1cat/K1m = 20.6 mM-1 s-1. Under conditions where the protein was in a monomeric state, the following parameters were determined for the splitting of bond 192-193 by integrating the Michaelis equation: k2cat = 0.056 s-1, K2m = 0.007 mM, and k2cat/K2m = 79.7 mM-1 s-1. Under the latter conditions the four enzymic reactions involved in the cleavage of bonds 189-190 and 192-193 were first-order reactions. The four corresponding apparent rate constants were calculated by using a computer program. Excellent agreement was obtained between concentrations of four molecular species measured during the reaction period and those calculated by using the four apparent rate constants.     

Kinetic studies of chicken and turkey liver mitochondrial aspartate aminotransferase.

The kinetic behaviour of chicken liver and turkey liver aspartate aminotransferases (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) was studied. Steady-state data were obtained from a wide range of concentrations of substrates and product L-glutamate. The data were fitted by rational functions of degree 1:1, 1:2 and 2:2 with respect to substrates and 0:1, 1:1, 0:2 and 1:2 with regard to product (L-glutamate), by using a non-linear regression program that guarantees the fit. The goodness of fit was improved by the use of a computer program that combines model discrimination parameter refinement and sequential experimental design. It was concluded that aspartate aminotransferase requires a minimum velocity equation of degree 2:2 for L-aspartate, 2:2 for 2-oxoglutarate and 1:2 for L-glutamate. Finally, a plausible kinetic mechanism that justifies these experimental results is proposed.     

Substrate characteristics of progesterone-albumin conjugates with 20β-hydroxysteroid dehydrogenase

Progesterone covalently conjugated to bovine serum albumin through the 21-position is a substrate for 20β-hydroxysteroid dehydrogenase (E.C.1.1.1.53) from S. hydrogenans. Varying the progesterone to albumin molar ratio in a range of 1:1 to 19:1 results in variations in the apparent Km and Vmax values. A maximum in substrate activity is obtained at a progesterone to albumin ratio of 6:1, while at either extreme in the range of these ratios the activity is a minimum. Progesterone-albumin conjugates attached at the steroid 2α-, 6β, or 11α-position in varying ratios did not produce measurable substrate activity. The results show that a steroid need not be free in solution to serve as a substrate for the steroid oxido-reductase.     

Derivation of a growth rate equation describing microbial surface colonization

A surface growth rate equation is derived which describes simultaneous growth and attachment during microbial surface colonization. The equation simplifies determination of attachment and growth rate, and does not require a computer program for solution. This rate equation gives the specific growth rate (Μ) as a function of the number of cells on the surface (N), the incubation period (t), and the number of colonies (C_i) containing either one cell, two cells, four cells, etc, as shown below. $$\mu = \frac{{\ln (\frac{N}{{C_i }} + 1)}}{t}$$ The attachment rate (A) is given by the following relationship: $$A = \mu C_i $$ The proposed colonization kinetics are compared with exponential growth kinetics using 3-dimensional computer plots. Colonization kinetics diverged most from exponential kinetics when the growth rate was low or the attachment rate was high. Using these kinetics, it is possible to isolate the effects of growth and attachment on microbial surface colonization.     

Crystal Structure of the Leucine Aminopeptidase from Pseudomonas putida Reveals the Molecular Basis for its Enantioselectivity and Broad Substrate Specificity

The zinc-dependent leucine aminopeptidase from Pseudomonas putida (ppLAP) is an important enzyme for the industrial production of enantiomerically pure amino acids. To provide a better understanding of its structure-function relationships, the enzyme was studied by X-ray crystallography. Crystal structures of native ppLAP at pH 9.5 and pH 5.2, and in complex with the inhibitor bestatin, show that the overall folding and hexameric organization of ppLAP are very similar to those of the closely related di-zinc leucine aminopeptidases (LAPs) from bovine lens and Escherichia coli. At pH 9.5, the active site contains two metal ions, one identified as Mn²⁺ or Zn²⁺ (site 1), and the other as Zn²⁺ (site 2). By using a metal-dependent activity assay it was shown that site 1 in heterologously expressed ppLAP is occupied mainly by Mn²⁺. Moreover, it was shown that Mn²⁺ has a significant activation effect when bound to site 1 of ppLAP. At pH 5.2, the active site of ppLAP is highly disordered and the two metal ions are absent, most probably due to full protonation of one of the metal-interacting residues, Lys267, explaining why ppLAP is inactive at low pH. A structural comparison of the ppLAP-bestatin complex with inhibitor-bound complexes of bovine lens LAP, along with substrate modelling, gave clear and new insights into its substrate specificity and high level of enantioselectivity.     

Kinetic study of the enzyme NADPH cytochrome-C (P-450) reductase: non-Michaelian behaviour

1. Contrary to what has been accepted until now, the enzyme exhibits non-Michaelian kinetics both against NADPH and against cytochrome-c as substrates; deviations were detected that have led to the proposition of a rate equation of minimum degree 2:2. 2. A general mechanism is proposed that includes, apart from the binding of the enzyme to NADPH, the formation of an enzyme-cytochrome-c complex, both routes leading to the formation of a ternary-complex NADPH-enzyme-acceptor. 3. From the latter, a series of intermediate steps finally leads to the release of the enzyme in conditions to start a new catalytic cycle. 4. Application of the King-Altman method to this mechanism yields a kinetic equation of degree 2:2 with respect to the cytochrome-c and NADPH, in accordance with our experimental results.     

Kinetic study of the inhibition of the amidolytic activity of thrombin by benzamidine and N-dansyl-(p-guanidino)-phenylalanine-piperidide (I-2581)

1. v([I]) data were obtained for the hydrolysis of the chromogenic substrate H-D-Phe-L-Pip-L-Arg-pNA (S-2238) by native human thrombin in the presence of the synthetic inhibitors Benzamidine and N-dansyl-(p-guanidino)-phenylalanine-piperidide (I-2581). v([S]) data were also obtained in the absence and presence of fixed concentrations of each of the inhibitors. 2. Analysis of the kinetic data was based on the numerical fitting to rate equations of the polynomial quotient type of degree n:m using nonlinear regression methods. The discrimination between rate equations with different degrees was performed by application of the statistical F test. 3. Of eight v([I]) curves fitted, in six cases it was found that degree 1:2 was significantly better than degree 1:1 at a confidence level of 95% or higher; in no case was a significant improvement found with rate equations with a higher number of parameters. For the v([S]) data, of eleven curves fitted it was found that in nine cases degree 2:2 significantly improved degree 1:1 at confidence levels 99% and in one case at a level of 95%; no significant improvement was found with rate equations of higher degree for these data either. 4. Our findings allow us to propose that inhibition of the amidolytic activity of native human thrombin by benzamidine and I-2581 may be accounted for by mechanisms whose v([I]) rate equation will be a minimum of degree 1:2, thus implying a pure inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)     

A comparison of DNA repair in cultured bovine lens epithelial cells and lung fibroblast cells

The effect of X-irradiation has been assessed on cultivated bovine lens epithelial cells by analysis of the DNA sedimentation in alkaline sucrose gradient and compared with bovine fibroblasts. (1) It can be concluded that for the same irradiation, DNA from both sources were broken to the same extent; (2) the rate of DNA repair was similar for both types of cells. The relation between these results and the fact that X-ray induces cataract very easily in the lens is discussed.     

Use of the F test for determining the degree of enzyme-kinetic and ligand-binding data. A Monte Carlo simulation study

1. Initial-rate data were simulated for 13 representative enzyme mechanisms with the use of several distributions of rate constants in order to locate conditions leading to v([S]) curves in physiological ranges of substrate concentration. 2. In all, 420 sets of such v([S]) curves were generated with the use of several choices of substrate concentration range (two, three or four orders of magnitude), number of experimental points (10, 15 or 20), error on v (5-10%) and standard deviation on v (5-9%) in order to simulate experimental results in a number of possible ways. 3. Curve-fitting was carried out to rational functions of degree 1:1, 2:2, . . ., 5:5 until there was no statistically significant decrease in the sum of weighted squared residuals as judged by the F test at 95% and 99% confidence levels. 4. It was checked whether the non-linear regression program had located a good minimum in the sum of squares by also fitting the data with the correct values of parameters as starting estimates. 5. A similar procedure was adopted with 110 sets of binding data simulated for 11 models, and the F test was used to see if fractional-saturation data generated by a binding polynomial of order n could be adequately fitted by one of order m, m less than n. 6. From the 530 simulations the F test was successful in fixing the correct degree with a probability of 0.62 at the 95% confidence level, but this fell with increase in degree as follows: 1:1 (0.98), 2:2 (0.71), 3:3 (0.43) and 4:4 (0.34), the first numbers being the degree of the rate equation and those in parentheses referring to the 95% confidence level. 7. It made little difference whether the 95% or the 99% confidence level was consulted, as there were very few borderline cases. 8. The chance of detecting deviations from Michaelis-Menten kinetics, i.e. terms of at least second-order in a rate equation of degree n:n, n greater than 1, was estimated to be about 0.8. 9. The probability of the F test leading to a spurious result due to error in the data was found to be about 0.04. 10. The probability with which 4:4 mechanisms can lead to v([S]) plots with no, one, two or three turning points was computed, and it was established that there is a small but finite chance that the increase in degree that occurs in some mechanisms when ES in equilibrium EP interconversions are explicitly allowed for can be detected by the F test.     

Steady-state kinetics of thyroid peroxidase. Evidence for a high degree rate equation using the F statistic

The initial-rate kinetics of bovine thyroid peroxidase are reported using 325 sets of concentrations of hydrogen peroxide and guaiacol. Extended ranges of concentrations are used and the v(S) profiles are fitted by rational functions of degree 2:2, 3:3 and +:4 by interactive non-linear regression analysis. Estimates of initial slopes in v(S) plots obtained by this regression are then replotted against the fixed substrate concentration and this confirms the need for a high-degree rate equation. Values of the F statistic indicate that the rate equation is 3:3 in guaiacol and 4:4 in hydrogen peroxide. It is concluded that the kinetics of peroxidase from bovine thyroid, like horse radish and human cervical mucus peroxidase and lactoperoxidase can be accommodated by the greater cyclic mechanism and that this is the minimal kinetic scheme for peroxidase In general.     

Structural studies on lens proteins

The sequence around the thiol group in lens proteins has been investigated. The proteins were converted into their carboxy[¹⁴C]methyl derivatives and submitted to partial acid hydrolysis, or digested with proteolytic enzymes. Acid hydrolysis of bovine α-crystallin gives N-seryl-(S-carboxymethyl)cysteine, Ser-CMCys (Waley, 1965a), but this dipeptide is not obtained from β-crystallin or γ-crystallin. Trypsin and chymotrypsin also give different peptides from the three crystallins. The radioactive peptide from α-crystallin and chymotrypsin has the sequence Ser-CMCys-Ser-Leu; another peptide, Asp-Leu-Leu-Phe, was also identified. The radioactive peptides obtained from bovine α-crystallin are probably also obtained from human α-crystallin, and from bovine and human albuminoid (the insoluble lens protein). α-Crystallin has been fractionated by chromatography in urea on DEAE-cellulose. Comparison of the fractions by peptide `mapping', and immunochemically, shows that they fall into two classes. The fraction eluted first differs from the later fractions, but the later fractions resemble each other The first fraction may represent impurities, or it may be a structurally different sub-unit of α-crystallin.     

Purification of long-chain acyl-CoA hydrolase from bovine heart microsomes and regulation of activity by lysophospholipids

Long-chain acyl-CoA hydrolase (EC 3.1.2.2) has been purified 12,000-fold from bovine heart muscle microsomes by extraction with Miranol detergent, followed by column chromatography on Reactive Blue agarose and DEAE-cellulose. The purified enzyme was nearly homogeneous on polyacrylamide gel electrophoresis and had a molecular weight of 41,000 in the presence of dodecyl sulfate. The specificity and kinetic properties of the enzyme were studied using several acyl-CoA derivatives as potential substrates. The enzyme showed a wide degree of specificity with little dependence on either the fatty acyl chain length or the degree of unsaturation of the acyl group. The kinetic properties were in accord with the Michaelis-Menten equation under most conditions, although high concentrations of substrates generally inhibited the enzyme. Arachidonoyl-CoA, which was the most effective substrate, had a K_m value of 0.4 μm and a V_max value of 6.0 μmol min⁻¹ mg⁻¹. The enzyme was strongly and specifically inhibited by lysophosphatidylcholine and lysophosphatidylinositol with kinetic inhibition constants of 16 and 30 nm, respectively. Other lysolipids and detergents such as deoxycholate and Triton X-100 were weak inhibitors. These properties and others distinguish this enzyme from other acyl-CoA hydrolases and support the idea that lysophospholipids may be important in vivo in the regulation of lipid metabolism.     

Does any enzyme follow the Michaelis—Menten equation?

Summary A literature search has been conducted to see to what extent steady-state kinetics studies in the period 1965–1976 have revealed deviations from Michaelis—Menten kinetics. It was found that over 800 enzymes have been reported as giving complex curves for a variety of reasons and a group by group classification of all these enzymes has been carried out listing all the types of variations reported and the authors' explanations. In addition, for highly complex curves, we have determined the minimum degree of the rate equation. There were very few determined attempts to demonstrate adherence to the Michaelis—Menten equation over a wide variety of experimental conditions and substrate concentration and almost invariably detailed experimental work revealed unsuspected complexities. For these reasons, it is concluded that the assumption that most enzymes follow the Michaelis—Menten equation can not be supported by an appeal to the literature.     

Differentiation of keratinocytesin vitro: a new culture vessel mimicking thein vivo situation

A culture vessel consisting of two independent chambers separated only by the growth substrate is described. Cells may be cultured on both sides of the growth substrate. Culture medium and gas exposure can independently be controlled in both compartments. Human hair follicles have been used as source of keratinocytes and the bovine eye lens capsule has been explored as growth substrate. The presence of 5% CO₂ in air in the lower compartment appears to have a significant effect on the morphology of the cultures. When the cultures are being exposed to air with 5% CO₂, the culture medium being applied in the lower compartment, formation of corneocytes characteristic for adult stratum corneum is induced, as evidenced by light and electron microscopy. To the knowledge of the authors, this stage of differentiationin vitro has not been obtained with previously described systems. Differentiation of the lower cell layers has been characterised with specific antibodies. The possible use of the system for applied and pure scientific research is discussed.     

Effect of helium on the respiration and glycolysis of mouse liver slices

It has been shown that helium has the ability to affect variously the rates of certain metabolic reactions in vitro as compared to nitrogen. An attempt has been made to approximate the sites of action in mouse liver preparations. The following results have been obtained by the substitution of a mixture of 80 per cent helium and 20 per cent oxygen for air: (a) An increase in the rate of oxygen consumption and carbon dioxide production to the same degree, the respiratory quotient remaining unchanged. (b) A decrease in the magnitude of cyanide inhibition. The effectiveness of helium increases with the degree of the cyanide inhibition. (c) No effect on the activity of slices which have been poisoned with fluoride when either lactate or pyruvate has been added as a substrate. (d) A change in the rate, and the slope of the curve of oxygen consumption in liver homogenates which are utilizing pyruvate as a substrate. The use of helium relative to nitrogen under anaerobic conditions causes: (a) A depression of the glycolytic rates in both mouse liver slices and diaphragm. (b) An increase in the carbon dioxide evolution and lactic acid production of mouse liver homogenates oxidizing either glucose and hexose diphosphate, or hexose diphosphate alone. In neither slices nor homogenates does the addition of fluoride and the use of pyruvate as the hydrogen acceptor alter the fundamental response of the preparations. The following hypotheses have been advanced and discussed in order to explain the observed phenomena: 1. Helium does not alter the substrate utilized by the tissue. 2. The gas interferes in some way with the cyanide-cytochrome oxidase bond, but may not affect cytochrome oxidase in the absence of cyanide. 3. The citric acid cycle is not subject to the influence of helium in tissue slices, but is altered in an unexplained fashion in homogenates. It is postulated that a rearrangement of particulate surfaces may be the significant factor here. 4. The glycolytic cycle is the site of both an inhibitory and an acceleratory effect of helium. The locus of the inhibition lies above the aldolase reaction and that of the acceleration between the aldolase and enolase reactions.