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The kinetics of enzyme deactivation provide useful insights on processes that determine the level of biological function of any enzyme. Photinus pyralis (firefly) luciferase is a convenient enzyme system for studying mechanisms and kinetics of enzyme deactivation, refolding, and denaturation caused by various external factors, physical or chemical by nature. In this report we present a study of luciferase deactivation caused by increased temperature (i.e., thermal deactivation). We found that deactivation occurs through a reversible intermediate state and can be described by a Transient model that includes active and reversibly inactive states. The model can be used as a general framework for analysis of complex, multiexponential transient kinetics that can be observed for some enzymes by reaction progression assays. In this study the Transient model has been used to develop an analytical model for studying a time course of luciferase deactivation. The model might be applicable toward enzymes in general and can be used to determine if the enzyme exposed to external factors, physical or chemical by nature, undergoes structural transformation consistent with thermal mechanisms of deactivation.

We derived an equation which describes the plot of the remaining enzyme activity versus ratio of initial concentration of suicide substrate to that of enzyme to obtain a partition ratio from the time-course of remaining enzyme activity. The simulation data calculated from the representative kinetic model for a suicide substrate were used to verify this equation, which approximated steady state kinetics. Although the time-dependent loss of enzyme activity is usually characterized by pseudo-first-order kinetics, the present results show that pseudo-first-order kinetics are followed only when the ratio of initial concentration of suicide substrate to that of enzyme is greater than the partition ratio. Our results also show that the present method can be used to obtain the partition ratio of a suicide substrate from the time-course of the remaining enzyme activity when the suicide substrate is given an arbitrary concentration of one, where the ratio of initial concentration of suicide substrate to that of enzyme is less than the partition ratio. The theoretically verified equation was also checked against reported experimental data for a microsomal enzyme system.

After incubation of 6α-hydroxyandrost-4-ene-3,17-dione with the aromatising enzyme system of human placenta, a 6-hydroxyoestrome was isolated which was identical with the compound formed after reduction of 6-oxo-oestradiol-17β with NaBH4 and subsequent selective oxidation with CrO3. When 6β-hydroxyandrost-4-ene-3,17-dione was incubated under the same conditions, a 6-hydroxyoestrone was isolated which was identical with the compound obtained after catalytic hydration of 6-oxo-oestrone. Since the configuration of the 6-hydroxyl groups in neutral steroids is well established, these experiments permit the definite assignment of configurations to the epimeric 6-hydroxylated phenolic steroids. The incubation of oestradiol-17β with rat-liver microsomes in the presence of NADPH2 and molecular oxygen led to the formation of 6α- and 6β- and hydroxyoestradiol-17β. The amount of 6α-hydroxyoestradiol-17β was about five times greater than that of 6β-hydroxyoestradiol-17β. When 6α- and 6β-hydroxyoestradiol-17β were incubated with rat-liver slices, 6-oxo-oestradiol-17β as well as 6α- and 6β-hydroxyoestrone were formed. Under the same conditions, 6α- and 6β-hydroxyoestrone were metabolised to 6-oxo-oestrone, 6α- and 6β-hydroxyoestradiol-17β. Incubation of 6-oxo-oestradiol-17β led to the formation of 6-oxo-oestrone, 6α- and 6β-hydroxyoestradiol-17β, whereas 6-oxo-oestrone yielded 6-oxo-oestradiol-17β, 6α- and 6β-hydroxyoestrone.

The authors have studied the influence of Ca++ and of Mn++ on the autolysis of trypsin; on the sensitivity to free trypsin of its enzymically inactive but “not denatured” derivatives, and finally on the action of trypsin on a substrate such that each enzyme molecule in play can only act once. The results obtained permit an explanation of the favouring action of Ca++ and of Mn++ on the enzymic activity of trypsin: when trypsin acts on a substrate, the concentration of the enzyme tends to decrease due to autolysis, a side reaction competing with hydrolysis of the substrate. Certain bivalent cations, especially Ca++ and Mn++, have an inhibiting action on autolysis, thus permitting a larger amount of enzyme to act on the substrate, these ions decreasing the susceptibility of trypsin to its own proteolytic action.

An enzyme system catalysing the reaction TPNH + GSSO3H ⇋ GSH + SO3H− + TPN+. has been partially purified from pea tissues. The characteristics of the enzyme have been studied.

The bacteriolytic system of a soil Actinomyces has been shown to consist of a bactericidal substance and a proteolytic enzyme system. The latter enzyme system lyses killed gram negative cells and killed gram positive cells rendered gram negative (with ribonucleinase), but has no action on the intact gram positive cells. Under the influence of the bactericidal substance, gram positive cells are killed and become gram negative. In this state they are susceptible to the action of the proteolytic enzymes.

1. 1. 8-Aminopurine is oxidised by mammalian xanthine oxidase along the following pathway: 8-Aminopurine→6-hydroxy-8-aminopurine→2,6-dihydroxy-8-aminopurine It is thus evident that oxidation of all three aminopurines takes a course different from the corresponding hydroxypurines. 2. 2. Adenine and 6-methylaminopurine are oxidised along the same pathway, but all other methyl derivatives of adenine are refractory. 3. 3. 2-Aminopurine is converted by a bacterial enzyme system into 2-amino-8-hydroxypurine. These results are discussed in terms of specific enzyme-substrate complexes.

A Pseudomonad which grows on hexane as sole carbon source was isolated from soil by enrichment-culture technique. Cell-free, soluble enzyme preparations of the organism catalyze the oxidation of 14C-labeled octane to radioactive octanoic acid. A pyridine nucleotide is required for this overall conversion in charcoal-treated bacterial extracts. n-Octanol and octaldehyde were identified as products of octane oxidation and the presence in the enzyme preparations of DPN-dependent dehydrogenases acting on octanol and octaldehyde was demonstrated.

A relatively stable purified enzyme preparation has been obtained from Rhodospirillum rubrum which catalyzes the formation of acetyl coenzyme A form ATP, acetate and CoA. AMP and pyrophosphate were shown to be end products of the reaction and also inhibitors of the reaction. The properties of the enzyme system have been described and found to be similar to those described for the yeast, heart muscle and plant enzymes.

At very high concentrations ( c ) of substrate the steady-state reaction rate for an enzyme obeying Michaelis-Menten kinetics approaches a maximum, k 2 E 0 , independent of c , while at low c the rate becomes proportional to c . Exact solutions for diffusion into a cylindrical cell containing a uniformly distributed Michaelis-Menten enzyme are obtained for both of these extreme cases. In addition on apprroximate solution has been found to the general non-linear equation, and procedures for obtaining the kinetic constants and diffusion parameters from experimental data are presented.

Bilirubin oxidase was immobilized to nylon fibres. A tri-enzyme system composed of glucose oxidase, bilirubin oxidase and horseradish peroxidase was also immobilized to the fibres. Both immobilized systems were tested and it was found that the latter gave enhanced oxidation rates for bilirubin.

A method for partial purification of an enzyme system from greening wheat leaves which converts 14C-labeled glutamate to delta-aminolevulinic acid is described. The purification entails the successive use of anion and cation exchange, followed by molecular sieving. The enzyme system is unstable in crude form, but the stability is markedly increased after column chromatography on CM-cellulose. The pH profile and the cofactor requirements suggest that at least two enzymes are involved.

The kinetic behavior of the heterogeneous sequential reaction, , has been studied. Both the forward and reverse inhibitions in the heterogeneous bienzyme system have a strong regulatory role over the reaction flux. Substrate inhibition in a heterogeneous monoenzyme system is less pronounced, due to diffusional constraints. The product inhibition in a heterogeneous monoenzyme system is manifested in a different manner, both qualitatively and quantitatively, from the free enzyme system. The pH-dependence of inhibition lends support to the hypothesis that fluctuations in the pH of the intracellular milieu can switch a required inhibition on or off.

Product inhibiton studies on yeast phosphoglycerate kinase (ATP:3-phospho-d-glycerate 1-phosphotransferase, EC 2.7.2.3) have been performed with 1,3-P2-glycerate. The results indicate that: 1. 1. The catalytic reaction can be affected via four substrate binding sites, two for MgATP2− and two for 3-P-glycerate. 2. 2. There is one catalytic centre per enzyme molecule. 3. 3. The catalytic reaction primarily occurs at the ‘first’ or ‘high affinity’ MgATP2− and 3-P-glycerate binding sites. The ‘second’ set of sub-sites for these substrates are located in a region for regulatin of the catalytic reaction. 4. 4. The products of the reaction, 1,3-P2-glycerate and ADP, are preferentially bound to the regulatory region. 5. 5. MgATP2− and 1,3-P2-glycerate are able to bind simultaneously to this region. When liganded with MgATP2− the apparent Ki value for 1,3-P2-glycerate increases from 3 μM to 20 μM.

The enhanced stability usually exhibited by enzymes after immobilization may be attributed either to a stabilization effect of the solid matrix on the bound enzyme molecule or to the influences of diffusional limitations on the observed activity. To allow the comparison of the intrinsic statilities of free and bound enzymes a simple graphical procedure for the removal of external diffusional effects on stability curves is described. It is based on the determination of substrate concentration differences between the enzyme micro- and microenvironment. Application of the method to aspartate aminotransferase bound to collagen membranes indicates that diffusional limitations for oxaloacetate are partly responsible for the observed stability enhancement. Comparison of the graphically obtained intrinsic profile with the stability curve of the soluble enzyme further demonstrate that the binding itself greatly increases the stability of aspartate aminotransferase.

The reduction of the A-ring of Δ 4 -3-ketosteriods by the rat ovary is carried out exclusively in the microsomal fraction, with the formation of 3β-hydroxy-5α-oriented metabolites. An enzyme system located in the soluble fraction is responsible for the conversion of these metabolites to their 3α-oriented isomers. No reversal of this epimerization could be demonstrated.

The effect of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) on the relative average oxygen yield per flash, ϕ(td) as a function of the time td between the flashes has been investigated in spinach chloroplasts. It was found, that at 2·10−7 M DCMU — where about 80% of the system II electron transport chains are blocked — the relative average oxygen yield per flash, ϕ(td), decreases with increasing time td. This effect shows that DCMU not only acts as an inhibitor of the reducing side of system II, but in addition accelerates the decay of the holes stored in the water-splitting enzyme system Y of photosynthesis.

Data are presented which confirm the presence of an active sulphate synthesizing enzyme system in cultivated connective tissues. Some of the properties of this enzyme system are described. Studies on the development of the granulomata show that the activity of the 3′-phosphoadenosine-5′-phosphosulphate synthesizing system is at maximum 96 h after the implantation of an ordinary cotton pellet and 48 h after the implantation of a cotton pellet containing carrageenin. It was found that pharmacologically high doses of cortisone inhibit the 3′-phosphoadenosine-5′-phosphosulphate synthesizing system both in ordinary granulomata and in those induced by carrageenin.

1. 1. Experiments designed to test the specificity of lipoproteins as inhibitors of glucose utilisation are presented. 2. 2. It has been shown that the difference between normal and diabetic plasma in regard to content of this material is largely quantitative. 3. 3. Lack of specificity is demonstrated as several enzymes other than those utilising glucose are inhibited. 4. 4. The possibility that the inhibitor acts by competing with organic phosphates for an active site on the enzyme molecule is discussed.

1. 1. A particle-free enzyme system of rat kidney has been prepared which is capable of decarboxylating GL and GLA. When fortified with UTP, TPP, DPN, ATP and Mg++ maximal effects are obtained. The reaction is aerobic. 2. 2. The enzyme system yields approximately 12% of CO2 which is predominantly derived from carbon-6 of GL and GLA. Increased yields may be obtained by further addition of enzyme.

Gastric juice from various animals contained an enzyme system capable of reducing the viscosity of a solution of native calf-skin collagen. These enzymes were confined in their action to the end-chains of collagen and could not be classified as collagenases. The end-chains contributed approx. 30% of the initial solution viscosity of collagen. Commercial preparations of pepsin (EC 3.4.4.1) differed in their ability to reduce the viscosity of collagen. Chromatographically homogeneous pepsin and three chromatographic subfractions (pepsins B, C and D) were demonstrated to have different actions on soluble collagen as indicated by viscosity changes. The individual pepsin enzymes had characteristic affinities for collagen and probably had different substrate specificities. The different actions of commercial pepsins on collagen could be accounted for by the presence of pepsin plus one or more of the more active subfractions. It was suggested that one sample of commercial crystalline pepsin and also human gastric juice contained a pepsin-like enzyme, other than any of the subfractions at present isolated, which was very much more active than any purified pepsin.

The effect of a number of 2-anilinothiophene derivates on the O2 yield per flash obtained by excitation with repetitive flashes and repetitive double flash groups has been investigated. It was found that: 1. 1. The decrease of the average O2 yield per flash at long times (td) between the flashes caused by the natural deactivation reactions of the watersplitting enzyme system is strongly accelerated by various derivatives of 2-anilinothiophene. 2. 2. The action of these agents as Acceleration of the Deactivation Reactions of the watersplitting Enzyme System Y (ADRY) substances is unequivocally proved. 3. 3. The ADRY effect is dependent on the acidity of the imino group, present in all ADRY agents so far known. These observations are discussed in connection with the mechanism of the activation—deactivation reactions of the watersplitting Enzyme System Y. A new class of photosynthesis effectors is established: the class of the ADRY agents. These substances provide a tool both to label and to modify the oxidizing equivalents which are responsible for the oxidation of water, in a similar way as the uncouplers can be used for the characterization of the high energy state in the phosphorylation process.

A procedure is described for the preparation from mammalian brain and kidney of an enzyme system which hydrolyses ATP to ADP and P 1 , and which requires both Mg 2+ , Na + and K + for full activity. The enzyme system prepared behaves qualitatively in its relations to cations and to g-strophanthin as the Mg 2+ + Na + + K + -activated, ATP-hydrolysing enzyme system previously isolated from peripheral crab nerves and red blood cells. The finding of this enzyme system in tissues from brain and kidney in which there is an active, linked transport of Na + and K + lends support to the assumption made on basis of the experiments with the enzyme system from peripheral nerves and from red blood-cell membranes that this enzyme system is involved in the active, linked transport of Na + and K + across the cell membrane.

Laccase of Rhus vernicifera was purified by column chromatography on an ion-exchange resin and zone electrophoresis. The purity was confirmed by sedimentation and electrophoresis. It was revealed that the enzyme molecule has a molecular weight of 120,000 and carries four atoms of cupric copper per molecule. The oxidation-reduction potential of the enzyme was measured spectrophotometrically in the presence of ferrocyanide-ferricyanide system. The E0′ value found was + 415 mV at 25° and pH 7.0. The purified enzyme showed two absorption maxima at 280 mμ and 615 mμ with a shoulder at 330 mμ.

An activity purified 21-fold from hematopoietic mouse spleen catalyzes the decarboxylaton of uroporphyrinogens I and III to stoichiometrically equivalent amounts of the corresponding coproporphyrinogens. Kinetic and stability studies indicate that the same enzyme system is involved in the two reactions. No partially decarboxylated intermediates are extracted with the reaction product.

A particulate enzyme system from the cell-free extract of Mycobacterium tuberculosis H37Ra catalyzes the reduction of palmityl-CoA to hexadecanol in the presence of ATP and NADPH. The hexadecanol product is esterified with endogenous acyl derivatives to form the wax esters. Two major radioactive wax ester products were identified as hexadecyl palmitate and hexadecyl stearate.

The kinetic behaviour of a matrix-bound three-enzyme system has been studied and compared with that of an analogous system consisting of the three enzymes unbound and in solution. The enzymes chosen were β-galactosidase, hexokinase and glucose-6-phosphate dehydrogenase which carry out three consecutive reactions. It was found that the efficiency of the coupled reaction catalyzed by the matrix-bound three-enzyme system was higher prior to reaching steady state than that catalyzed by the corresponding soluble system. Although a higher efficiency than that of the corresponding soluble system was also found for the coupled reaction when only the two last enzymes of the matrix-bound system were implicated, the measured increase in efficiency between the complete three-enzyme systems was even more pronounced, indicating a cumulative efficiency effect.

Further studies on the liver microsomal 6-hydroxylation of lipid-soluble 3-substituted indoles demonstrate that the responsible enzyme system possesses many features in common with the microsomal drug metabolizing enzymes. However, the hydroxylation is not inhibited by bipyridyl, and animal pretreatment with phenobarbital exerts a weak stimulatory effect on the yield of 6-hydroxy compounds. The enzyme is not highly stereospecific as the d - and l -forms of N -acetyltryptophan are hydroxylated at equal rates. 6-Fluoro and methyl groups are not displaceable by hydroxy groups. Non-enzymatic hydroxylation of indolic substrates by chemical model systems gives rise to more than one monohydroxy product; on the other hand, microsomal hydroxylation produces only the 6-isomer, indicating positional specificity. microsomal hydroxylation produces only the 6-isomer, indicating positional specificity. Comparison with aniline p -hydroxylase shows that the two systems are different. The mechanism of the reaction is discussed.

1. 1. The intact utilization of 1-monopalmitin by intestinal mucosal enzymes for the synthesis of triglycerides has been demonstrated. 2. 2. The enzyme does not demonstrate a specificity for the d or l isomer 3. 3. The first acylation of 1-monopalmitin occurs at the corresponding pirmary hydroxy group forming the 1,3-diglyceride intermediate. 4. 4. The subcellular location and optimal requirements of the enzyme system catalyzing the acylation have been determined.