Chitin and chitinase: A kinetic model

A model is described for the action of insect molting chitinase on chitin microfibrils in cuticle. The model reconciles the disparate structures proposed for chitin in the literature. It also accounts for the kinetic characteristics of molting fluid chitinase insofar as known from in vitro studies, viz. positive co-operativity of possibly three catalytic sites, complexity, and processivity. These have hitherto been difficult to account for in vivo, given the arrangement of chitin in anhydrous microfibrils in arthropod cuticle.     

Characterization of ligand-induced states of maize homoserine dehydrogenase

The threonine-sensitive homoserine dehydrogenase (L-homoserine: NAD(P)+ oxido-reductase), isolated from seedlings of Zea mays L., is characterized by variable kinetic and regulatory properties. Previous analysis of this enzyme suggested that it is capable of ligand-mediated interconversions among four kinetically distinct states (S. Krishnaswamy and J. K. Bryan (1983) Arch. Biochem. Biophys. 222, 449-463). These forms of the enzyme have been identified and found to differ in oligomeric configuration and conformation. In the presence of KCl and threonine a rapid equilibrium among three species of the enzyme (B, T, and K) is established. Each of these species can undergo a unique slow transition to a steady-state form under assay conditions. Results obtained from gel-filtration chromatography and sucrose density centrifugation indicate that the B and steady-state forms are tetramers and the T and K states are dimers. Evidence is presented to indicate that the rapid conversion from one dimeric species to the other can only occur via formation of the tetrameric B state. Chromatography under reacting-enzyme conditions provides direct support for the slow formation of a common steady-state species from any one of the other forms of the enzyme. The rate of transition is influenced by threonine, homoserine, NAD+, and, for transitions involving association reactions, by enzyme concentration. Small, reproducible differences in the apparent size of the T and K forms, and the B and steady-state species, are attributed to changes in conformation. This conclusion is supported by differential susceptibility of the enzymic states to proteolytic inactivation, by different rates of inactivation by dithio-bis-nitrobenzoate, and by alterations in their thermal stability. In addition, the B, T, and K states of the enzyme exhibit unique intrinsic fluorescence spectra. Spectral changes are shown to closely parallel changes in kinetic and hysteretic properties of the enzyme. The results of diverse methods of analysis are internally consistent, and provide considerable support for the conclusion that this pleiotropic regulatory enzyme can exist in any of several physically distinct states.     

New human liver alcohol dehydrogenase forms with unique kinetic characteristics

All adult and infant human liver homogenates studied thus far show two previously unreported forms of alcohol dehydrogenase on starch gel electrophoresis. Under the conditions employed, these forms migrate toward the anode and readily stain for pentanol but virtually not for ethanol oxidizing activity. In contrast, all human ADH isoenzymes identified previously are cathodic and react equally well with either substrate. These new ADH forms have been separated from the other known ones by DEAE-cellulose chromatography and are then purified on Agarose-hexane-AMP. Although the physical characteristics of the new anodic ADH forms are similar to those of the known human ADH isoenzymes, the former are not inhibited by 12 mM 4-methyl pyrazole, oxidize ethanol very poorly and appear to prefer longer chain alcohols as substrates.     

Purification and steady-state kinetic analysis of yeast thiosulfate reductase.

Thiosulfate reductase has been purified approximately 70-fold from an extract of bakers' yeast. An enzyme with a molecular weight of 17,000, a Stokes radius of 19 Å, and a pI of 5.1 was obtained. Initial velocity and inhibition studies indicate that the substrates add in a random fashion. Further evidence suggests that the rapid-equilibrium assumption is not totally applicable. The enzyme has two distinct but closely situated substrate binding sites—one for compounds with an RSO₃^? structure and one for the sulfhydryl substrate.     

Hemoglobin Constant Spring: hemoglobin synthesis in heterozygous and homozygous states.

Thirteen adult and one newborn heterozygotes, and three homozygotes for hemoglobin Constant Spring were examined for globin chain synthesis. Reticulocytes from venous blood were incorporated with [³H]-leucine in an incubation mixture for 3 hours. Globin prepared from the radioactive, washed red cells was fractionated by CM-cellulose chromatography in 8 M urea and the total radioactivity of each globin chain was determined. The mean of $\text{α}\text{β}$ ratio in the heterozygotes was 1.34 ± SD 0.08, which is significantly different from that of 1.07 ± SD 0.03 in eleven normal controls. The $\text{α}\text{β+γ}$ ratio in the heterozygous neonate was also 1.39. The $\text{α}\text{β}$ ratios in the three homozygotes were around 1.6. The α-Constant Spring chain appears to be over produced, but it may be unstable or labile, not fully available for conjugation with the non alpha chains.     

Human liver cytosolic malate dehydrogenase: Purification,kinetic properties,and role in ethanol metabolism

Cytosolic malate dehydrogenase from human liver was isolated and its physical and kinetic properties were determined. The enzyme had a molecular weight of 72,000 ± 2000 and an amino acid composition similar to those of malate dehydrogenases from other species. The kinetic behaviour of the enzyme was consistent with an Ordered Bi Bi mechanism. The following values (μm) of the kinetic parameters were obtained at pH 7.4 and 37 °C: K_a, 17; K_ia, 3.6; K_b, 51; K_ib, 68; K_p, 770; K_ip, 10,700; K_q, 42; K_iq, 500, where a, b, p, and q refer to NADH, oxalacetate, malate, and NAD⁺, respectively. The maximum velocity of the enzyme in human liver homogenates was 102 μmol/min/g wet wt of liver for oxalacetate reduction and 11.2 μmol/min/g liver for malate oxidation at pH 7.4 and 37 °C. Calculations using these parameters showed that, under conditions in vivo, the rate of NADH oxidation by the enzyme would be much less than the maximum velocity and could be comparable to the rate of NADH production during ethanol oxidation in human liver. The rate of NADH oxidation would be sensitive to the concentrations of NADH and oxalacetate; this sensitivity can explain the change in cytosolic $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ redox state during ethanol metabolism in human liver.     

Catalytic and kinetic properties of purified high-affinity cyclic AMP phosphodiesterase from dog kidney

High-affinity cyclic AMP phosphodiesterase purified to homogeneity from dog kidney was studied with respect to its stability, its catalytic and kinetic properties, and its sensitivity to pharmacological agents. The enzyme was shown to rapidly lose activity upon dilution to low protein concentrations in aqueous media, but this activity loss was largely prevented by the presence of bovine serum albumin or ethylene glycol. Similarly, maximum activity required bovine serum albumin to be present during incubation for activity analysis. Enzyme activity required a divalent cation; Mg²⁺, Mn²⁺, and Co²⁺ each supported activity, but highest activity was obtained with Mg². The temperature optimum ranged from 30 to 45 °C and depended on substrate concentration; the E_a = 10,600 cal/mol. The pH optimum of the enzyme was broad, with a maximum from pH 8.0 to 9.5. The enzyme exhibits linear Michaelis-Menton kinetics for hydrolysis of cyclic AMP at all substrate concentrations tested and for hydrolysis of cyclic GMP at > 20 μm. The K_m for cyclic AMP hydrolysis was 2 μm, and that for cyclic GMP hydrolysis was 312 μm. The K_i values for the competitive inhibition of hydrolysis of each substrate by the other were similar to their K_m values suggesting a single active site. Cyclic AMP hydrolysis was weakly inhibited by cyclic GMP, cyclic IMP, adenine, and adenosine, but was not inhibited by the mono-, di, or trinucleotides of adenosine, guanosine, or inosine. Activity was competitively inhibited with K_i values in the micromolar range by drugs representative of methylxanthines, isoquinolines, pyrazolopyridines, imidazolidinones, triazolopyrimidines, pyridylethylenediamines, phenothiazines, and calcium antagonists. The results are discussed with reference to the similarities and differences between high- and low-affinity phosphodiesterase forms.     

Microcomputer interface for computer-assisted enzyme kinetic studies with uv-vis spectrophotometers

A simple microcomputer interface, consisting of only three integrated chips, for the input of digitized data from a uv-vis spectrophotometer is described. The interface multiplexes four binary-coded decimal digits, four possible decimal point positions, and an algebraic sign into a single eight-bit parallel input port of the microcomputer. The interface design is also adaptable in a general fashion to any analytical instrument which can present digitized data, with the expansion capability of accommodating more than the number of digits and decimal positions noted in this specific example. Software developed specifically to drive the interface and several general applications to computer-assisted kinetic studies are discussed.     

Effect of internal diffusion in heterogeneous enzyme systems: Evaluation of true kinetic parameters and substrate diffusivity

The effect of internal diffusion on the overall reaction rate in spherical particles and membranes containing immobilized enzymes has been investigated theoretically. Since they represent open systems, the MichaelisMenten kinetics is obeyed in the absence of diffusional effects at steady state even at high enzyme concentrations. When internal diffusion perturbs the reaction, the system can not be described any more by K_M and V_max? alone, but is conveniently characterized by the modulus. Assuming that only internal diffusion interferes with the enzyme reaction, the effect of the modulus on the overall rate of reaction is illustrated by the results of computer calculations. Plots of the overall reaction rate against the substrate concentration are hyperbolas at various moduli for both membranes and spherical particles and no sigmoidal curves are obtained with immobilized enzyme systems. Since the conventional plots of enzyme kinetics do not yield straight lines under such conditions, a graphical method is proposed to determine K_M and V_max? as well as the substrate diffusivity in the enzymic medium.     

S-adenosylhomocysteine hydrolase from hamster liver: Purification and kinetic properties

3-Deazaadenosine is both an inhibitor of and a substrate for S-adenosylhomocysteine hydrolase. Its administration to rats results in the accumulation of both S-adenosylhomocysteine and 3-deazaadenosylhomocysteine in the liver and other tissues. In hamsters, however, the administration of 3-deazaadenosine results only in the accumulation of 3-deazaadenosylhomocysteine (P. K. Chiang and G. L. Cantoni (1979) Biochem. Pharmacol. 28, 1897). In order to investigate the possible reasons for this difference, S-adenosylhomocysteine hydrolase from hamster liver has been purified to homogeneity and some of its kinetic and physical parameters have been determined. The molecular weight of the native enzyme is 200,000 with a subunit molecular weight of 48,000. The K_m's for adenosine and 3-deazaadenosine are about 1.0 μm, and the V_max's are also similar. The K_m for S-adenosylhomocysteine is 1.0 μm, or more than 10 times smaller than the K_m of the rat liver enzyme. This difference in K_m value may explain the differences in the response of rat and hamster liver to the administration of 3-deazaadenosine. S-Adenosylhomocysteine hydrolase from hamster liver exhibits an interesting kinetic property in that its activity can be affected bimodally by either adenosine or adenosine Anal.ogs. At very low concentrations of these analogs, the activity of S-adenosylhomocysteine hydrolase can be stimulated by 10–30%, and at higher concentrations these same analogs become competitive inhibitors.     

A kinetic analysis of d-xylose transport in rhodotorula glutinis

The kinetics of D-xylose transport were studied in Rhodotorula glutinis. Analysis of the saturation isotherm revealed the presence of at least two carriers for d-xylose in the Rhodotorula plasma membrane. These two carriers exhibited Km values differing by more than an order of magnitude. The low K_m carrier was repressed in rapidly growing cells and depressed by starvation of the cells.Several hexoses were observed to inhibit d-xylose transport. In the studies reported here, the inhibitions produced by d-galactose and 2-deoxy-d-glucose were examined in some detail in order to define the interactions of these sugars with the d-xylose carriers. 2-Deoxy-d-glucose competitively inhibited both of the d-xylose carriers. In contrast, only the low-K_m carrier was competitively inhibited by d-galactose.     

Alteration of thermal stability of glucose oxidase associated with the redox states

By the method of differential scanning calorimetry, it was found that thermal stability of glucose oxidase was dependent on its redox states. The oxidized form showed an apparent denaturation temperature at 76°C and the denaturation enthalpy was approximately 865 kcal/mol. On reduction of the enzyme, the denaturation temperature increased by about 10°, but no significant change was seen in the denaturation enthalpy. The activation energies of the denaturation of the oxidized and the reduced enzymes were about 89 and 103 kcal/mol, respectively. These results may imply conformational changes in the catalytic turnover of this enzyme.     

Homoserine kinase of Escherichia coli: kinetic mechanism and inhibition by L-aspartate semialdehyde

The kinetic mechanism of homoserine kinase, purified to homogeneity from Escherichia coli, was examined by initial velocity techniques at pH 7.6. Whereas ATP displayed normal Michaelis-Menten saturation kinetics (Km = 0.2 mM), L-homoserine showed hyperbolic saturation kinetics only up to a concentration of 0.75 mM (Km = 0.15 mM). Above this concentration, L-homoserine caused marked but partial inhibition (Ki approximately 2 mM). The kinetic data indicated that the addition of substrates to homoserine kinase occurs by a preferred order random mechanism, with ATP preferentially binding before L-homoserine. When the ATP concentration was varied at several fixed inhibitory concentrations of L-homoserine, the resulting inhibition pattern indicated hyperbolic mixed inhibition. This suggested a second binding site for L-homoserine. L-Aspartate semialdehyde, an amino acid analog of L-homoserine, proved to be an alternative substrate of homoserine kinase (Km = 0.68 mM), and was subsequently used as a probe of its kinetic mechanism. In aqueous solution, at pH 7.5, this analog was found to exist predominantly (ca 85%) as its hydrated species. When examined as an inhibitor of the physiological reaction, L-aspartate semialdehyde showed mixed inhibition versus both L-homoserine and ATP. Although the pH profiles for the binding of L-homoserine as a substrate (Km) and as an inhibitor (Ki) were identical, the kinetic data were best fit to a two-site model, with separate catalytic and inhibitory sites for L-homoserine.     

Competitive inhibition by the D-isomer in racemic mixtures used as substrate in kinetic studies: a simple method for data treatment

A set of equations was applied that allows the use of racemic mixtures for the estimation of kinetic parameters in systems where the L-isomer is substrate and the D-isomer a competitive inhibitor, displaying data as double reciprocal plots. A statistical treatment was introduced that renders compatible the output of presently available programs with the specific model requirements, with few additional calculations. An example is shown with beta-trypsin and its inhibition of benzozyl-D-arginine p-nitroanilide (Ki = 1.12 mM, pH 8,0, 37 degrees C).     

Daily alterations in plasma testosterone in boars at different ages

Eighteen purebred Yorkshire boars, reared outdoors on concrete, were randomly assigned in equal numbers to three age groups (150 +/- 7, 200 +/- 7 and 250 +/- 7 days of age) for the purpose of examining endogenous testosterone concentrations over a 24-hr period. Blood was obtained at 30-min intervals for 24 hr, and plasma testosterone concentrations were quantitated by radioimmunoassay. For each set of 24-hr samples, mean concentration (C), frequency (F) and magnitude (M) of secretory spikes of testosterone were determined. There was no difference (P>.10) in C, F and M across the three age groups. When averaged over ages, testosterone mean (+/- SEM) for C, F and M was 1.4 +/- .1 ng/ml, 2.9 +/- .5 and 3.0 +/- .3 ng/ml, respectively. The results suggest that testosterone secretory patterns are established in boars by five months of age and that these patterns may be influenced by degree of daylinght.     

A comparison of the denatured states of alpha-lactalbumin and lysozyme

We have carried out a study of the denaturation of bovine α-lactalbumin by LiClO₄, LiCl, GuCl and urea, using difference spectroscopy, viscometry, polarimetry, and optical rotatory dispersion. These denaturants give rise to three different denatured states (GuCl and urea give the same state), which cannot be related to each other as members of a simple linear progression from the native state to the completely disordered state. The data require that LiClO₄ unfolds one part of the molecule, LiCl another, and GuCl or urea the whole molecule. There are striking parallels between the denaturation behaviour of lactalbumin and earlier observations on hen egg-white lysozyme, a protein with which it is about 40% homologous, and we believe the mass of the data supports the hypothesis, advanced by Browne et al. (1969), that the two proteins have similar backbone conformations.     

Human lysosomal beta-glucosidase: kinetic characterization of the catalytic, aglycon, and hydrophobic binding sites

Three binding sites on highly purified lysosomal beta-glucosidase from human placenta were identified by studies of the effects of interactions of various enzyme modifiers. The negatively charged lipids, taurocholate and phosphatidylserine, were shown to be noncompetitive, nonessential activators of 4-methylumbelliferyl-beta-D-glucoside hydrolysis. Similar results were observed using the natural substrate, glucosyl ceramide, and low concentrations of taurocholate (less than 1.8 mM) or phosphatidylserine (0.5 mM). However, higher concentrations resulted in a complex partial inhibition of glucosyl ceramide hydrolysis. Increasing concentrations of phosphatidylserine obviated the effects of taurocholate, suggesting that these compounds compete for a common binding site on the enzyme. Glucosyl sphingosine and its N-hexyl derivative were potent noncompetitive inhibitors of the enzyme activity using either substrate. Taurocholate (or phosphatidylserine) and glucosyl sphingosine were shown to be mutually exclusive, indicating competition for a common binding site. In contrast, octyl- and dodecyl-beta-glucosides were linear-mixed-type inhibitors of glucosyl ceramide or 4-methylumbelliferyl-beta-D-glucoside hydrolysis, indicating at least two binding sites on the enzyme. Inhibition by these alkyl beta-glucosides was observed only in the presence of taurocholate or phosphatidylserine. The competitive component [Ki (slope)] for the two alkyl beta-glucosides decreased with increasing alkyl chain length, and was unaffected by increasing taurocholate or phosphatidylserine concentration. The noncompetitive component [Ki (intercept)] was nearly identical for both alkyl beta-glucosides and was decreased by increasing taurocholate or phosphatidylserine concentration. These results indicated that the negatively charged lipids and alkyl beta-glucosides were not mutually exclusive, but interacted with different binding sites on the enzyme. Gluconolactone was shown to protect the enzyme from inhibition by the catalytic site-directed covalent inhibitor, conduritol B indicating an interaction at a common binding site. In the presence of substrate, taurocholate facilitated the inhibition of gluconolactone or conduritol B epoxide. These studies indicated that lysosomal beta-glucosidase had at least three binding sites: (i) a catalytic site which cleaves the beta-glucosidic moiety, (ii) an aglycon site which binds the acyl or alkyl moieties of substrates and some inhibitors, and (iii) a hydrophobic site which interacts with negatively charged lipids and facilitates enzyme catalysis.     

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides: revised kinetic mechanism and kinetics of ATP inhibition

The kinetic mechanisms of the NAD- and NADP-linked reactions catalyzed by glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides were examined using product inhibition, dead-end inhibition and alternate substrate experiments. The results are consistent with a steady-state random mechanism for the NAD-linked and an ordered, sequential mechanism with NADP+ binding first for the NADP-linked reaction. Thus, the enzyme can bind NADP+, NAD+, and glucose 6-phosphate, but the enzyme-glucose 6-phosphate complex can react only with NAD+, not with NADP+. This affects the rate equation for the NADP-linked reaction by introducing a term for a dead-end enzyme-glucose 6-phosphate complex. The kinetic mechanisms represent revisions of those proposed previously (C. Olive, M.E. Geroch, and H.R. Levy, 1971, J. Biol. Chem. 246, 2047-2057) and provide a kinetic basis for the regulation of coenzyme utilization of the enzyme by glucose 6-phosphate concentration (H.R. Levy, and G.H. Daouk, 1979, J. Biol. Chem. 254, 4843-4847) and NADPH/NADP+ concentration ratios (H.R. Levy, G.H. Daouk, and M.A. Katopes, 1979, Arch, Biochem. Biophys. 198, 406-413). The kinetic mechanisms were found to be the same at pH 6.2 and pH 7.8. The kinetics of ATP inhibition of the NAD- and NADP-linked reactions were examined at pH 6.2 and pH 7.8. The results are interpreted in terms of ATP addition to binary enzyme-coenzyme and enzyme-glucose 6-phosphate complexes.     

alpha-Galactosidase (melibiase) from Saccharomyces carlsbergensis: structrual and kinetic properties.

This report describes structural and kinetic properties of the purified α-galactosidase from Saccharomyces carlsbergensis. This galactosidase has many similar properties to other exocellular enzymes in yeast which have been reported. Its molecular weight of 300,000 is comparable; it has similar carbohydrate content (57%) and amino acid and carbohydrate composition. That is, 35% of its amino acid residues can be accounted for by threonine, serine, and aspartic acid. Its carbohydrate composition is primarily mannose (90–95%) with approximately 7% glucose and 1% glucosamine. The enzyme is very stable to both acidic and alkaline conditions as well as to heating to 50 °C. α-Galactosidase remains active after incubation with as much as 1% sodium dodecyl sulfate at 30 °C. However, the enzyme is denatured with urea and guanidine hydrochloride. The loss of activity is proportional to the urea concentration, the nondenatured enzyme being responsible for the remaining activity. Inactivation by urea is partially reversible. With urea or 60 °C heat denaturation, the enzyme dissociates into two types of subunits as revealed by polyacrylamide gel electrophoresis with sodium dodecyl sulfate. Thus, α-galactosidase is the first external enzyme from yeast in which an oligomeric structure is reported. The enzyme catalyzes the hydrolysis of p-nitrophenyl-α-d-galactoside, melibiose, and raffinose with similar pH optima and V_max. However, the affinity is 20-fold lower for raffinose than for the other two substrates. Sugars having the same configuration in carbons 2, 3, and 4 as galactose competitively inhibit the enzyme.