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R T Wedding

Publications and source records attributed to R T Wedding.

At least 55 records · Page 3Linked to original sources
PubMed1982

Kinetic Ramifications of the Association-Dissociation Behavior of NAD Malic Enzyme : A Possible Regulatory Mechanism.

NAD malic enzyme can exist in dimer, tetramer, or octamer form. Freshly prepared enzyme from Solanum tuberosum var. Chieftan exists predominantly as the octamer and during storage is progressively converted into lower molecular weight forms. High ionic strength favors dimer formation, whereas high concentrations of malate or citrate favor tetramer formation. The tetramer is the most active form, having a low K(m) for malate and a high V(max). The dimer, with its high K(m) and low V(max), is the least active form. Malate may regulate NAD malic enzyme by controlling its state of oligomerization.

Journal Article↗
PubMed1981

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.

Journal Article↗
PubMed1980

Regulation of the 2-oxoglutarate dehydrogenase lipoate succinyltransferase complex from cauliflower by nucleotide. Pre-steady state kinetics and physical studies.

Analysis of the binding of thiamin pyrophosphate to the 2-oxoglutarate dehydrogenaselipoate succinyltransferase multienzyme complex using pre-steady state kinetic methods revealed that the presence of 2-oxoglutarate is not necessary for binding, although it does stabilize the complex by slowing the rate of dissociation of the holoenzyme. The rate of binding of thiamin-PPi to the enzyme and the subsequent enzyme activation are not limited by a reaction at C-2 of the thiazolium ring of thiamin-PPi since no kinetic isotope effect is observed when 2-D-thiamin-PPi is substituted for the protonated cofactor. The presence of 5'-AMP, which activates the reaction producing both a V and a Km response, causes a significant increase in kon for thiamin-PPi. The AMP analog 1,N6-ethenoadenosine-5'-monophosphate (epsilon-AMP) also activates the reaction, but shows only a K effect, with no influence on V. This effector reduces Kd for the thiamin-PPi2-oxoglutarate dehydrogenase complex by increasing kon. The change in kon for thiamin-PPi in response to changes in hydrogen ion concentration shows pK values which are unaffected by the addition of AMP, in this respect resembling the steady state kinetic response of V/Km and differing from the pH profile of V. The dissociation constant of holoenzyme is relatively insensitive to pH over the range pH 6 to 9, but in the presence of AMP the Kd, which is decreased in the range from pH 7 to 8, increases sharply at higher or lower pH values.

Acyltransferases↗
PubMed1977

Overall mechanism and rate equation for O-acetylserine sulfhydrylase.

Saturation curves for O-acetylserine sulfhydrylase using the substrate analogues O-propionyl-L-serine, O-butyryl-L-serine, and beta-chloro-L-alanine all exhibit substrate inhibition and yield Km values comparable to O-acetyl-L-serine, except the O-butyryl derivative which has a Km 5-fold higher. Since all analogues are used as substrates and yield similar kinetic parameters in most cases, it is possible that they share a common intermediate. This evidence also suggests that specificity of O-acetylserine sulfhydrylase resides in the fact that the beta-substituted moiety on L-serine is a good leaving group. The overall rate equation for O-acetylserine sulfhydrylase was derived. A comparison of the numerical integration of the rate equation and an experimental time course is given.

Alanine↗
PubMed1977

Cystine content of legume seed proteins: estimation by determination of cysteine with 2-vinylquinoline, and relation to protein content and activity of cysteine synthase.

The cystine content of the protein of a number of different lines of legume seeds has been determined by the method of Krull et al. which selectively reacts cysteine residues of intact, reduced proteins with 2-vinylquinoline, giving an adduct with an absorption maximum at 318 nm. Some seed lines were found to have 3.5 times as much cysteine as the seed line with the lowest cysteine content, perhaps offering opportunities for improvement in the nutritional quality of bean seed proteins through breeding and selections. While no correlation between cysteine levels and protein content was observed, a positive correlation was found between the specific activity of the terminal enzyme of cysteine synthesis, cysteine synthase, and the cysteine content of seeds.

Cysteine↗
PubMed1977

Purification and initial kinetic characterization of different forms of o-acetylserine sulfhydrylase from seedlings of two species of phaseolus.

Purification of O-acetylserine sulfhydrylase (OASS) from seedlings of two species of Phaseolus reveals the presence in both species of two forms of this enzyme. The isolation and purification procedure gives purification of 7- to 160-fold for individual isoenzymes with specific activities ranging from 33 IU mg(-1) to 775 IU mg(-1) protein.Detailed study of the basic kinetic parameters of the OASS isoenzymes indicates that both forms from Phaseolus vulgaris (which are of about equal specific activity) display substrate inhibition by S(2-) above 1 mm and positive cooperativity at lower concentrations of S(2-). With respect to O-acetylserine (OAS), the second substrate of the reaction, one P. vulgaris isoenzyme shows substrate inhibition by OAS concentrations above 10 mm, while the second is unaffected by OAS concentrations up to 50 mm. The isoenzymes from Phaseolus polyanthus (one of which has a specific activity 24 times higher than the other) are slightly and approximately equally inhibited by both S(2-) and OAS.

Journal Article↗
PubMed1976

A reaction mechanism from steady state kinetic studies for O-acetylserine sulfhydrylase from Salmonella typhimurium LT-2.

It has been determined from steady state kinetic studies using the sulfide ion selective electrode that O-acetylserine sulfhydrylase catalyzes a Bi Bi Ping Pong reaction between O-acetyl-L-serine and sulfide. Both O-acetyl-L-serine (OAS) and sulfide exhibit strong competitive substrate inhibition. A fit of all the data to the equation for the mechanism yields KOAS = 0.149 +/- 0.059 mM and KIOAS = 46.91 +/- 10.06 mM for O-acetyl-L-serine and KS2- = 0.066 +/- 0.004 mM and KIS2- = 0.013 +/- 0.006 mM for sulfide. Product inhibition studies varying either substrate at changing fixed levels of cysteine demonstrate that cysteine combines with enzyme at two places along the reaction sequence to produce inhibition with KiCys = 1.048 +/- 0.048 mM and KICys = 11.4 +/- 0.5 mM. Relatively high concentrations of acetate are required to produce inhibition and at least part of the acetate inhibition is due to ionic strength. However, the ability of acetate to reverse the spectral shift produced from the binding of O-acetyl-L-serine to enzyme and the isotope exchange between [14C]acetate and O-acetyl-L-serine does demonstrate that the O-acetyl-L-serine to acetate half-reaction is reversible. There is some doubt as to the specificity of acetate as a product inhibitor, since propionate can also be used to reverse the spectral shift. Spectral studies using ths spectral shift produced from binding O-acetyl-L-serine to enzyme confirms the assignment of a ping-pong mechanism since the spectral intermediate produced is alpha-aminoacrylic acid in Schiff base with pyridoxal phosphate and, therefore, the acetyl moiety has been beta eliminated. Isotope exchange has been demonstrated for both the O-acetyl-L-serine to acetate and sulfide to cysteine half-reactions which also confirms a ping-pong mechanism.

Cysteine↗
PubMed1976

Malate Dehydrogenase and NAD Malic Enzyme in the Oxidation of Malate by Sweet Potato Mitochondria.

Over a range of concentrations from less than 0.1 mm to more than 70 mm, sweet potato root mitochondria display a bimodal substrate saturation isotherm for malate. The high affinity portion of the isotherm has an apparent Km for malate of 0.85 mm and fits a rectangular hyperbolic function. The low affinity portion of the isotherm is sigmoid in character and gives an apparent S(0.5) of 40.6 mm and a Hill number of 3.7.Extracts of sweet potato mitochondria contain both malate dehydrogenase and NAD malic enzyme. The malate dehydrogenase, assayed in the forward direction at pH 7.2, shows typical Michaelis-Menten kinetics with a Km for malate of 0.38 mm. The NAD malic enzyme shows pronounced sigmoidicity in response to malate with a Hill number of 3.5 and an S(0.5) of 41.6 mm.On the basis of the normal kinetics, the Km, and the fact that oxaloacetate production from malate by mitochondria appears most active at low malate concentrations, the high affinity portion of the malate isotherm with mitochondria is attributed to malate dehydrogenase. The low affinity portion of the malate isotherm with mitochondria is thought, on the basis of the similarity of S(0.5) values, the Hill numbers, and the greater production of pyruvate from malate at high malate concentrations, to represent the activity of the NAD malic enzyme.

Journal Article↗
PubMed1972

Malate dehydrogenase isoenzymes from cotton leaves: molecular weights.

Malate dehydrogenase isolated from leaves of the cotton plant (Gossypium hirsutum L.) appears in the form of several isoenzymes. Four of the isoenzymes found in cotton leaf extracts appear to be charge isomers with a molecular weight of approximately 60,000. A fifth malate dehydrogenase isoenzyme found in leaf extracts has a molecular weight of approximately 500,000. Under appropriate conditions it is possible to form this high molecular weight isoenzyme from at least one of the smaller isoenzymes. In addition, malate dehydrogenase isoenzymes of approximately 700,000 and 130,000 molecular weight have been observed under some conditions, although these isoenzymes do not appear in the crude cotton leaf preparations. The relationship of this heterogeneity with respect to size and to the discrepancies in the number and size of malate dehydrogenase isoenzymes reported from plant tissues may be significant.

Journal Article↗