The kinetics of enzyme-catalyzed reactions with two or more substrates or products☆III. Prediction of initial velocity and inhibition patterns by inspection

Ww, Cleland

doi:10.1016/0006-3002(63)91816-x

Cited by 203 publications

(90 citation statements)

References 3 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Glutamate was a classic noncompetitive inhibitor towards 2-oxoglutarate, whereas aspartate functioned in the same way towards oxaloacetate. These observations are consistent with a Ping Pong Bi Bi mechanism (8). The inhibition constant of the enzymeglutamate complex of root nodule fraction II (24 ± 3 mM, Table II) was approximately 3 times larger than the values estimated for the root nodule fraction I (8.3 ± 1 mM) and that of the root fraction (6.9 + 0.…”

Section: Kinetic Properties Of Got Present In Different Fractions Of supporting

confidence: 87%

Glutamate Oxaloacetate Transaminase in Pea Root Nodules

Appels¹,

Haaker²

1991

Plant Physiol.

View full text Add to dashboard Cite

Glutamate oxaloacetate transaminase (L-glutamate:oxaloacetate aminotransferase, EC 2.6.1.1 [GOT]), a key enzyme in the flow of carbon between the organic acid and amino acid pools in pea (Pisum sativum L.) root nodules, was studied. By ion exchange chromatography, the presence of two forms of GOT in the cytoplasm of pea root nodule cells was established. The major root nodule form was present in only a small quantity in the cytoplasm of root cells. Fractionation of root nodule cell extracts demonstrated that the increase in the GOT activity during nodule development was due to the increase of the activity in the cytoplasm of the plant cells, and not to an increase in activity in the plastids or in the mitochondria. The kinetic properties of the different cytoplasmic forms of GOT were studied. Some of the Km values differed, but calculations indicated that not the kinetic properties but a high concentration of the major root nodule form caused the observed increase in GOT activity in the pea root nodules. It was found that the reactions of the malate/aspartate shuttle are catalyzed by intact bacteroids, and that these reactions can support nitrogen fixation. It is proposed that the main function of the nodule-stimulated cytoplasmic form of GOT is participation in this shuttle.

show abstract

Section: Kinetic Properties Of Got Present In Different Fractions Of supporting

confidence: 87%

Glutamate Oxaloacetate Transaminase in Pea Root Nodules

Appels¹,

Haaker²

1991

Plant Physiol.

View full text Add to dashboard Cite

show abstract

“…K d values can then be determined by fitting the data to a hyperbolic decay curve, or to the quadratic form of the binding isotherm if binding is very tight relative to enzyme concentration: (10) …”

Section: Fluorescence Approaches To Studying Adenylation and Aminoacymentioning

confidence: 99%

“…The advantages of this approach are that (i) it does not require large amounts of materials; (ii) the assays are generally fast and involve minimal workup; and (iii) data from a large number of enzyme and tRNA variants can be rapidly obtained and compared by determining the ratio of (k cat /K m ) cognate /(k cat /K m ) non-cognate [9]. Although steady state kinetic assays do not allow elementary steps to be characterized, initial velocity and product inhibition patterns can provide information on the orders of substrate binding and product release [10]. The most important steady state kinetic assays for the aminoacyl-tRNA synthetases are the pyrophosphate exchange assay, which measures the rate of exchange of [ 32 P]-PP i into ATP, and the aminoacylation assay, which measures the rate of AA-tRNA AA formation.…”

Section: Introductionmentioning

confidence: 99%

Methods for kinetic and thermodynamic analysis of aminoacyl-tRNA synthetases

Francklyn

First

Perona

et al. 2008

Methods

107

120

View full text Add to dashboard Cite

The accuracy of protein synthesis relies on the ability of aminoacyl-tRNA synthetases (aaRSs) to discriminate among true and near cognate substrates. To date, analysis of aaRSs function, including identification of residues of aaRS participating in amino acid and tRNA discrimination, has largely relied on the steady state kinetic pyrophosphate exchange and aminoacylation assays. Pre-steady state kinetic studies investigating a more limited set of aaRS systems have also been undertaken to assess the energetic contributions of individual enzyme-substrate interactions, particularly in the adenylation half reaction. More recently, a renewed interest in the use of rapid kinetics approaches for aaRSs has led to their application to several new aaRS systems, resulting in the identification of mechanistic differences that distinguish the two structurally distinct aaRS classes. Here, we review the techniques for thermodynamic and kinetic analysis of aaRS function. Following a brief survey of methods for the preparation of materials and for steady state kinetic analysis, this review will describe pre-steady state kinetic methods employing rapid quench and stopped-flow fluorescence for analysis of the activation and aminoacyl transfer reactions. Application of these methods to any aaRS system allows the investigator to derive detailed kinetic mechanisms for the activation and aminoacyl transfer reactions, permitting issues of substrate specificity, stereochemical mechanism, and inhibitor interaction to be addressed in a rigorous and quantitative fashion.

show abstract

“…This observation indicates that CPZ binds to the Na ϩ -free, E 2 form. The ratio K 0.5 (Na ϩ )/EP max (12,13) increased Ͼ6-fold, indicating direct interaction of CPZ with Na ϩ binding. We also showed that CPZ competes with oligomycin for NKA (SI).…”

mentioning

confidence: 96%

Capsazepine, a synthetic vanilloid that converts the Na,K-ATPase to Na-ATPase

Mahmmoud

2008

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Capsazepine (CPZ), a synthetic capsaicin analogue, inhibits ATP hydrolysis by Na,K-ATPase in the presence but not in the absence of K ؉ . Studies with purified membranes revealed that CPZ reduced Na ؉ -dependent phosphorylation by interference with Na ؉ binding from the intracellular side of the membrane. Kinetic analyses showed that CPZ stabilized an enzyme species that constitutively occluded K ؉ . Low-affinity ATP interaction with the enzyme was strongly reduced after CPZ treatment; in contrast, indirectly measured interaction with ADP was much increased, which suggests that composite regulatory communication with nucleotides takes place during turnover. Studies with lipid vesicles revealed that CPZ reduced ATP-dependent digitoxigenin-sensitive 22 Na ؉ influx into K ؉ -loaded vesicles only at saturating ATP concentrations. The drug apparently abolishes the regulatory effect of ATP on the pump. Drawing on previous homology modeling studies of Na,K-ATPase to atomic models of sarcoplasmic reticulum Ca-ATPase and on kinetic data, we propose that CPZ uncouples an Na ؉ cycle from an Na ؉ /K ؉ cycle in the pump. The Na ؉ cycle possibly involves transport through the recently characterized Na ؉ -specific site. A shift to such an uncoupled mode is believed to produce pumps mediating uncoupled Na ؉ efflux by modifying the transport stoichiometry of single pump units.sodium pump ͉ uncoupling ͉ potassium occlusion N a,K-ATPase (NKA), a P-type cation pump (1), utilizes energy from ATP hydrolysis to exchange intracellular Na ϩ for extracellular K ϩ against their electrochemical potential gradients. The functional pump contains an ␣-subunit that undergoes substrateinduced conformational transitions, coupling ATP hydrolysis to ion transport, and a ␤-subunit important for maturation and targeting of the complex to the membrane (2).According to the Albers-Post scheme, occlusion of three intracellular Na ϩ ions promotes phosphorylation of the ␣-subunit from ATP, whereas occlusion of two extracellular K ϩ ions promotes dephosphorylation (2). Depending on the specific ionic composition on both sides of the membrane, the pump catalyzes several partial reactions (3). In the absence of K ϩ , NKA mediates ATP-driven Na ϩ transport processes, including exchange as well as uncoupled efflux (reviewed in ref. 4). Furthermore, in inside-out vesicles of human red cells, the ratio of net 22 Na ϩ influx/ 86 Rb ϩ efflux was reduced at sub-millimolar cytoplasmic Na ϩ concentrations. This finding indicates that under this particular condition, the pump can function with less than a maximum number of sites filled with Na ϩ ions (5).In sarcoplasmic reticulum (SR) Ca 2ϩ -ATPase (SERCA), the best characterized P-type pump, ATP hydrolysis can take place without Ca 2ϩ transport to allow for thermogenesis (6). This uncoupling is prominent in the presence of a Ca 2ϩ gradient across the SR membrane. Recent studies have shown that sarcolipin, a small SERCA regulator, increases heat production by this pump (7). This finding demonstrates a regulatory inter...

show abstract

The kinetics of enzyme-catalyzed reactions with two or more substrates or products☆III. Prediction of initial velocity and inhibition patterns by inspection

Cited by 203 publications

References 3 publications

Glutamate Oxaloacetate Transaminase in Pea Root Nodules

Glutamate Oxaloacetate Transaminase in Pea Root Nodules

Methods for kinetic and thermodynamic analysis of aminoacyl-tRNA synthetases

Capsazepine, a synthetic vanilloid that converts the Na,K-ATPase to Na-ATPase

Contact Info

Product

Resources

About