Kinetic studies of rat liver hexokinase D (glucokinase) in non-co-operative conditions show an ordered mechanism with MgADP as the last product to be released

The kinetic mechanism of rat liver hexokinase D ('glucokinase') was studied under non-co-operative conditions with 2-deoxyglucose as substrate, chosen to avoid uncertainties derived from the co-operativity observed with the physiological substrate, glucose. The enzyme shows hyperbolic kinetics with respect to both 2-deoxyglucose and MgATP2-, and the reaction follows a ternary-complex mechanism with Km = 19.2±2.3mM for 2-deoxyglucose and 0.56±0.05mM for MgATP2-. Product inhibition by MgADP- was mixed with respect to MgATP2- and was largely competitive with respect to 2-deoxyglucose, suggesting an ordered mechanism with 2-deoxyglucose as first substrate and MgADP- as last product. Dead-end inhibition by N-acetylglucosamine, AMP and the inert complex CrATP [the complex of ATP with chromium in the 3+ oxidation state, i.e. Cr(III)—ATP], studied with respect to both substrates, also supports an ordered mechanism with 2-deoxyglucose as first substrate. AMP appears to bind both to the free enzyme and to the E·dGlc complex. Experiments involving protection against inactivation by 5,5′-dithiobis-(2-nitrobenzoic acid) support the existence of the E·MgADP- and E·AMP complexes suggested by the kinetic studies. MgADP-, AMP, 2-deoxyglucose, glucose and mannose were strong protectors, supporting the existence of binary complexes with the enzyme. Glucose 6-phosphate failed to protect, even at concentrations as high as 100mM, and MgATP2- protected only slightly (12%). The inactivation results support the postulated ordered mechanism with 2-deoxyglucose as first substrate and MgADP- as last product. In addition, the straight-line dependence observed when the reciprocal value of the inactivation constant was plotted against the sugar-ligand concentration supports the view that there is just one sugar-binding site in hexokinase D.

Time-dependent inactivation of chick-embryo prolyl 4-hydroxylase by coumalic acid. Evidence for a syncatalytic mechanism

From the structure-activity relationships of known competitive inhibitors, coumalic acid (2-oxo-1,2H-pyran-5-carboxylic acid) was deduced to be a potential syncatalytic inhibitor for chick-embryo prolyl 4-hydroxylase. The compound caused time-dependent inactivation, the reaction rate being first-order. The inactivation constant was 0.094 min-1, the Ki 17 mM and the bimolecular rate constant 0.09 M-1 X S-1. Human prolyl 4-hydroxylase and chick embryo lysyl hydroxylase were also inactivated, though to a lesser extent. Inactivation could be prevented by adding high concentrations of 2-oxoglutarate or its competitive analogues to the reaction mixture. In Lineweaver-Burk kinetics, coumalic acid displayed S-parabolic competitive inhibition with respect to 2-oxoglutarate. The inactivation reaction had cofactor requirements similar to those for the decarboxylation of 2-oxoglutarate. Enzymic activity was partially preserved in the absence of iron, but the rescue was incomplete, owing to decreased stability of the enzyme under this condition. Coumalic acid also decreased the electrophoretic mobility of the alpha-subunit, but the beta-subunit was not affected. Prolonged incubation of coumalic acid above pH 6.8 led to loss of its inactivating potency, owing to hydrolysis. It is concluded that the inactivation of prolyl 4-hydroxylase by coumalic acid is due to a syncatalytic mechanism. The data also suggest that the 2-oxoglutarate-binding site of the enzyme is located within the alpha-subunit.

AN INDIRECT KINETIC METHOD FOR ESTIMATING THE AFFINITY BETWEEN HEPARIN AND HEPARIN COFACTOR II

The heparin-dependent inactivation of alpha-thrombin by heparin cofactor II was studied in buffer media with a pH ranging from 6 to 9 and an ionic strength from 0.05 M to 0.80 M. We used a heparin fraction with a mean Mr of 16,000 .The log dose response curves (logarithm of the 2nd order inactivation constant vs. the logarithm of heparin concentration) display a sigmoidal behavior. The lower and upper limiting plateau and the steepness of the ascending limb are characteristic for every pH and ionic strength. Similar log dose response curves can be observed for the heparin-mediated inactivation of factor Xa and plasmin by ATIII,indicating that enhancement of the inhibition only depends on heparin-inhibitor binding despite the presence of heparin-enzyme complexes.This type of inactivation mechanism is clearly different from the one observed for the thrombin- and factor IXa - ATIII interactions which are characterized by a maximum in the log dose response curves.Therefore we can make the assumption that heparin-inhibitor binding is of major importance in the heparin-mediated thrombin-HCII interaction.Our experimental data were fit to a model which describes the dependence of the observed inactivation constant upon the concentration of heparin-HCII complex.This complex concentration is a function of the initial heparin and HCII concentrations and the dissociation constant K0 of the heparin-HCII complex.The model allows the estimation of K0 in various buffer media.A decrease of pK0 with increasing buffer concentration can be observed.The upper limiting plateau value for kobs which is often referred to as the intrinsic activity of heparin also decreases with increasing ionic strength.At pH 7 and 8 and an ionic strength of 0.4 M we-found K0 values of 1.00E-07 M.At pH 6 and an ionic strength of 0.8 M Kq eguals 4.00E-04 M indicating a markedly decreased affinity of heparin for HCII.Through a detailed analysis of the pH profile for K0 we might gain insight in the nature of the binding sites for heparin on the inhibitor.

Inactivation of TEM-1 β-lactamase by 6-acetylmethylenepenicillanic acid

6-Acetylmethylenepenicillanic acid is a new kinetically irreversible inhibitor of various beta-lactamases. Interaction between 6-acetylmethylenepenicillanate and purified TEM-1 beta-lactamase during the inactivation process was investigated. 6-Acetylmethylenepenicillanate inhibited the enzyme in a second-order fashion with a rate constant of 0.61 microM-1 . S-1. The apparent inactivation constant decreased in the presence of increasing concentrations of the substrate benzylpenicillin. Native enzyme (pI 5.4) was converted into two inactive forms with pI 5.25 and 5.15, the latter form being transient and readily converted into the more stable form with pI 5.15. Even a 50-fold excess of inhibitor over enzyme did not produce any other inactivated species of the enzyme. All the results obtained suggest that 6-acetylmethylenepenicillanate is a potent irreversible and active-site-directed inhibitor of TEM-1 beta-lactamase.

Calcium efflux from squid axons under constant sodium electrochemical gradient.

The effect of varying Nao and Nai on Ca efflux while maintaining the ratio Nao/Nai constant was explored in squid giant axons dialyzed with and without ATP. In the absence of ATP, the Ca efflux increased 3.4 +/- 0.2-fold when the Nao/Nai concentrations were reduced from 440/80 to 110/20 mM. In the presence of ATP a similar change did not have an appreciable effect. The inhibition of Ca efflux produced by Nai was studied in the presence and in the absence of ATP. In the absence of ATP, inhibition is very marked and is reminiscent of a unimolecular noncompetitive reaction (inactivation constant [KI] of 34 +/- 5 mM of Nai) whereas in the presence of ATP, the slight inhibition observed indicates that ATP probably increases the KI to 200mM. From the inhibition of the Ca efflux produced by Nai in the presence or absence of ATP a curve describing the dependence of Nai of the ATP-promoted fraction of Ca efflux was constructed. The effect of Nao on the Ca efflux was studied as a function of [Na]i: at low Nai, an activation constant (KA) of 41 mM for Nao was obtained either in the presence of in the absence of ATP. As the intracellular Na is increased in the presence of ATP, Nai seems to have no effect on the apparent half-activation constant. However, in the absence of ATP, the KA for activation increases along a sigmoid curve reaching a value of 112 mM at 100 mM Nai. It is concluded that the Ca efflux system uses the energy of the Na electrochemical gradient. The action of Nai appears to be such that the interaction of a single Na+ is sufficient to block Ca extrusion whereas several Naps externally are necessary to activate Ca extrusion.

THE KINETICS OF THE DECOMPOSITION OF PEROXIDE BY CATALASE

It has been shown that the experimental results obtained by Morgulis in a study of the decomposition of hydrogen peroxide by liver catalase at 20°C. and in the presence of an excess of a relatively high concentration of peroxide are quantitatively accounted for by the following mechanisms. 1. The rate of formation of oxygen is independent of the peroxide concentration provided this is greater than about 0.10 M. 2. The rate of decomposition of the peroxide is proportional at any time to the concentration of catalase present. 3. The catalase undergoes spontaneous monomolecular decomposition during the reaction. This inactivation is independent of the concentration of catalase and inversely proportional to the original concentration of peroxide up to 0.4 M. In very high concentrations of peroxide the inactivation rate increases. 4. The following equation can be derived from the above assumptions and has been found to fit the experiments accurately. See PDF for Equation in which x is the amount of oxygen liberated at the time t, A is the total amount of oxygen liberated (not the total amount available), and K is the inactivation constant of the enzyme.