Carbon-13 and deuterium isotope effects on the reaction catalyzed by glyceraldehyde-3-phosphate dehydrogenase.

Carbon-13 and deuterium isotope effects have been measured on the reaction catalyzed by rabbit muscle glyceraldehyde-3-phosphate dehydrogenase in an effort to locate the rate-limiting steps. With D-glyceraldehyde 3-phosphate as substrate, hydride transfer is a major, but not the only, slow step prior to release of the first product, and the intrinsic primary deuterium and 13C isotope effects on this step are 5-5.5 and 1.034-1.040, and the sum of the commitments to catalysis is approximately 3. The 13C isotope effects on thiohemiacetal formation and thioester phosphorolysis are 1.005 or less. The intrinsic alpha-secondary deuterium isotope effect at C-4 of the nicotinamide ring of NAD is approximately 1.4; this large normal value (the equilibrium isotope effect is 0.89) shows tight coupling of hydrogen motions in the transition state accompanied by tunneling. With D-glyceraldehyde as substrate, the isotope effects are similar, but the sum of commitments is approximately 1.5, so that hydride transfer is more, but still not solely, rate limiting for this slow substrate. The observed 13C and deuterium equilibrium isotope effects on the overall reaction from the hydrated aldehyde are 0.995 and 1.145, while the 13C equilibrium isotope effect for conversion of a thiohemiacetal to a thioester is 0.994, and that for conversion of a thioester to an acyl phosphate is 0.997. Somewhat uncertain values for the 13C equilibrium isotope effects on aldehyde dehydration and formation of a thiohemiacetal are 1.003 and 1.004.

Kinetic properties of NAD malic enzyme from cauliflower.

The kinetic characteristics of NAD malic enzyme purified to homogeneity from cauliflower florets have been examined. Free NAD+ is the active form of this coenzyme. Double-reciprocal plots of data obtained by varying NAD+ and malate2- at a saturating concentration of Mg2+ or by varying Mg2+ and NAD+ at a saturating level of malate2-are of intersecting type. This indicates that NAD malic enzyme obeys a sequential mechanism. Analysis of these sets of data suggests that each of these substrate pairs binds randomly to the enzyme. However, each substrate binds tighter when others are already present on the enzyme. NAD malic enzyme cannot decarboxylate malate2- in the absence of either Mg2+ or NAD+. Arrhenius plots of the NAD-linked reaction are concave downward, indicating the existence of two rate-determining steps with activation energies of 26.5 and 14.2 kcal/mol, respectively. In addition to Mg2+, the enzyme can also use Mn2+ and Co2+. Using Co2+ in place of Mg2+ does not change Vmax or Km, malate2- but the Km for metal and NAD+ are greatly decreased. At pH 7.0 and above, Mn2+ isotherms and malate2- curves with Mn2+ are nonlinear and appear to be composed of two separate saturation curves. NAD malic enzyme is completely and irreversibly inactivated by N-ethylmaleimide. The enzyme is also irreversibly inactivated approximately 50% by KCNO.

Allosteric regulation of the NAD malic enzyme from cauliflower: activation by sulfate.

Activation of the NAD malic enzyme by sulfate has been found to be due only to free, uncomplexed SO42-; the complex of sulfate with divalent cations has no measurable effect on the enzyme. Activation by SO42- is shown to result from a decrease in the Km for malate2-. Thus, activation is observed only at less than saturating levels of this substrate. The interactions of NAD+ and Mg2+ with the enzyme are not affected by SO42-. Response of the activity of the enzyme to SO42- is biphasic in that the activation seen at low SO42- concentrations is overcome as the level of the effector is increased so that at very high SO42- concentrations, activation disappears. This deactivation process is not simply a reversal of the activation mechanism; instead, it involves a decrease in the intrinsic Vmax of the reaction. The response to SO42- is also affected by the presence of other anionic effectors of the malic enzyme. Fumarate2- and phosphate are shown to directly affect the activation process by increasing the affinity of the enzyme for SO42-. While Cl- does not greatly affect the extent of stimulation, it does inhibit the enzyme without reducing the activated rate so that the apparent percentage activation over the control is very large, due to the lowered control rate. In contrast to the sensitivity of the malic enzyme reaction to pH, activation by SO42- appears to be independent of H+ concentration. The possibility that sulfate is a physiological effector of this and other plant mitochondrial enzymes is discussed.

Allosteric regulation of the NAD malic enzyme from cauliflower: activation by fumarate and coenzyme A.

Activation of the NAD malic enzyme is shown to be caused by free, uncomplexed fumarate2-. Mg-fumarate has no detectable effect on the enzyme. Fumarate2- isotherms are biphasic in that they consist of an activating as well as a deactivating region. Activation is shown to result from an increase in the affinity of the enzyme for malate2- while deactivation results from a reduction in Vmax. Phosphate does not affect the response of the enzyme to fumarate2-, while Cl- inhibits the enzyme in a manner that cannot be overcome by fumarate2-. SO42-, another activator of the malic enzyme, reduces the Ka for fumarate2- from 3.9 to 2.1 mM. Activation of the enzyme by coenzyme A (CoA) is hyperbolic with a Ka for CoA of 2.1 microM. Fumarate2- reduces this value to 1.2 microM. CoA, like SO42-, is able to increase the affinity of the enzyme for fumarate2-, decreasing its Ka by 56%. An additional effect of fumarate2- is to cause the interconversion of different catalytic forms of the enzyme which exist when Mg2- is limiting. On the basis of these results, a model of the number and types of allosteric sites present on the NAD malic enzyme is proposed.

Slow Transients in the Activity of the NAD Malic Enzyme from Crassula.

The NAD malic enzyme from Crassula argentea shows a slow reaction transient in the form of a lag before reaching a steady-state rate in assays. This lag, which has a half-time or tau ranging from seconds to many minutes under various conditions, poses problems in the interpretation of kinetic data with this enzyme. The NAD malic enzyme from Kalanchoë daigremontiana has a similar lag.The lag is greatest in freshly prepared enzyme and diminishes with storage at -70 degrees C, but the activity of the enzyme also diminishes with storage.The lag is inversely proportional to the concentration of enzyme, both in the assay and in storage prior to assay. The lag is also inversely proportional to the concentration of malate used in the assay, which poses particular problems because the lag with low malate concentrations may be so long that activity begins to be lost before the steady-state rate is reached.Various buffer ions produce different lags, but the lag with all buffers is longer than in the absence of buffer. The effectors CoA and SO(4) (2-) in the assay substantially decrease the lag. The lag is shorter with Mn(2+) as the required divalent cation than when Mg(2+) is used.The response of enzyme activity to pH shows that the intrinsic activity is greater with magnesium than with manganese, although the rate actually attained is lower with Mg(2+) because the pK values for the response to pH are closer together when that cation is used. The enzyme has a higher optimum pH and a broader response to pH when Mn(2+) is used. The change in lag with pH follows the general pattern of activity with longer lags at intermediate pH values.Preincubation of the enzyme with various reaction components and effectors reduces the lag, with NADH being the most effective. The presence of NADH in the assay is much more effective, but none of the treatments tried will completely eliminate the lag of freshly prepared enzyme.

Statistical package for analysis of competition ELISA results.

A computation and analysis program has been assembled to facilitate the use of competition ELISA and similar assays for studies of antigen regulation and turnover. The program fits sigmoidal standard curves using a 4-parameter logistic function, determines amounts of antigen from the equation which defines the standard curve, and calculates specific activities by linear regression of the levels of antigen in varying amounts of total protein. An optional weighting function is provided to adjust for systematic non-uniform variance. Outlying points are identified during the linear regression, and the user may delete them or redefine the acceptable working range of the standard curve. The program provides a complete print-out of the data and optional plots of the fitted standard curve and the regression analysis of the samples, as well as statistics which are useful for quality control. It also provides the option of storing the data points from the standard curve on magnetic diskettes. The package is written in BASIC for a Wang Model 2200 computer equipped with a magnetic diskette drive, line printer, and flat-bed X-Y plotter, but it is readily adaptable to other systems and input/output devices.