Kinetics of the Quaternary Structure Change of Aspartate Transcarbamylase Triggered by Succinate, a Competitive Inhibitor

Biochemistry ◽  
1994 ◽  
Vol 33 (33) ◽  
pp. 10007-10012 ◽  
Author(s):  
Hirotsugu Tsuruta ◽  
Patrice Vachette ◽  
Takayuki Sano ◽  
Michael F. Moody ◽  
Yoshiyuki Amemiya ◽  
...  
Keyword(s):  
Quaternary Structure ◽  
Competitive Inhibitor ◽  
Structure Change ◽  
Aspartate Transcarbamylase ◽  
Kinetics Of

scholarly journals Aspartate-90 and arginine-269 of hamster aspartate transcarbamylase affect the oligomeric state of a chimaeric protein with an Escherichia coli maltose-binding domain

1998 ◽  
Vol 329 (2) ◽  
pp. 243-247 ◽  
Author(s):  
Yu QIU ◽  
N. Jeffrey DAVIDSON
Keyword(s):  
Escherichia Coli ◽  
Structural Integrity ◽  
Quaternary Structure ◽  
Binding Domain ◽  
Carbamoyl Phosphate ◽  
Oligomeric State ◽  
Aspartate Transcarbamylase ◽  
Catalytic Subunits ◽  
E Coli ◽  
Kinetics Of

Residues Asp-90 and Arg-269 of Escherichia coli aspartate transcarbamylase seem to interact at the interface of adjacent catalytic subunits. Alanine substitutions at the analogous positions in the hamster aspartate transcarbamylase of a chimaeric protein carrying an E. coli maltose-binding domain lead to changes in both the kinetics of the enzyme and the quaternary structure of the protein. The Vmax for the Asp-90 → Ala and Arg-269 → Ala substitutions is decreased to 1/21 and 1/50 respectively, the [S]0.5 for aspartate is increased 540-fold and 826-fold respectively, and the [S]0.5 for carbamoyl phosphate is increased 60-fold for both. These substitutions decrease the oligomeric size of the protein. Whereas the native chimaeric protein behaves as a pentamer, the Asp-90 variant is a trimer and the Arg-269 variant is a dimer. The altered enzymes also exhibit marked decreases in thermal stability and are inactivated at much lower concentrations of urea than is the unaltered enzyme. Taken together, these results are consistent with the hypothesis that both Asp-90 and Arg-269 have a role in the enzymic function and structural integrity of hamster aspartate transcarbamylase.

Influence of Nucleotide Effectors on the Kinetics of the Quaternary Structure Transition of Allosteric Aspartate Transcarbamylase

2005 ◽  
Vol 348 (1) ◽  
pp. 195-204 ◽  
Author(s):  
Hiro Tsuruta ◽  
Hiroshi Kihara ◽  
Takayuki Sano ◽  
Yoshiyuki Amemiya ◽  
Patrice Vachette
Keyword(s):  
Quaternary Structure ◽  
Aspartate Transcarbamylase ◽  
Structure Transition ◽  
Kinetics Of

Kinetic Aspects of the Interaction of Blood Clotting Enzymes

1968 ◽  
Vol 19 (03/04) ◽  
pp. 364-367 ◽  
Author(s):  
H. C Hemker ◽  
P. W Hemker
Keyword(s):  
Enzyme Kinetics ◽  
Blood Clotting ◽  
Competitive Inhibition ◽  
Competitive Inhibitor ◽  
Kinetics Of

SummaryThe enzyme kinetics of competitive inhibition under conditions prevailing in clotting tests are developed and a method is given to measure relative amounts of a competitive inhibitor by means of the t — D plot.

scholarly journals Mitochondrial adenosine triphosphatase from human placenta--inhibition by free magnesium ions of ITP hydrolysis.

1994 ◽  
Vol 41 (1) ◽  
pp. 39-44 ◽  
Author(s):  
Z Aleksandrowicz
Keyword(s):  
Competitive Inhibition ◽  
Competitive Inhibitor ◽  
Hill Coefficient ◽  
Negative Cooperativity ◽  
Magnesium Ions ◽  
Beef Liver ◽  
Bicarbonate Ions ◽  
The Hill ◽  
Kinetics Of ◽  
Free Magnesium

The effects of Mg2+ and bicarbonate on the kinetics of ITP hydrolysis by soluble ATPase (F1) from human placental mitochondria were studied. Increasing amounts of Mg2+ at fixed ITP concentration, caused a marked activation of F1 followed by inhibition at higher Mg2+ concentration. The appropriate substrate for the mitochondrial F1 seems to be the MgITP complex as almost no ITP was hydrolysed in the absence of magnesium. Mg2+ behaved as a competitive inhibitor towards the MgITP complex. In this respect the human placental enzyme differ from that from other sources such as yeast, beef liver or rat liver. The linearity of the plot presenting competitive inhibition by free Mg2+ of MgITP hydrolysis (in the presence of activating bicarbonate anion) suggests that both Mg2+ and MgITP bind to the same catalytic site (Km(MgITP) = 0.46 mM, Ki(Mg) = 4 mM). When bicarbonate was absent in the ITPase assay, placental F1 exhibited apparent negative cooperativity in the presence of 5 mM Mg2+, just as it did with MgATP as a substrate under similar conditions. Bicarbonate ions eliminated the negative cooperativity with respect to ITP (as the Hill coefficient of 0.46 was brought to approx. 1), and thus limited inhibition by free Mg2+. The results presented suggest that the concentration of free magnesium ions may be an important regulatory factor of the human placental F1 activity.

scholarly journals The kinetics of transport of lactate and pyruvate into isolated cardiac myocytes from guinea pig. Kinetic evidence for the presence of a carrier distinct from that in erythrocytes and hepatocytes

1989 ◽  
Vol 264 (2) ◽  
pp. 409-418 ◽  
Author(s):  
R C Poole ◽  
A P Halestrap ◽  
S J Price ◽  
A J Levi
Keyword(s):  
Guinea Pig ◽  
Cardiac Myocytes ◽  
Ventricular Myocytes ◽  
Silicone Oil ◽  
Free Acid ◽  
Competitive Inhibitor ◽  
Isolated Cardiac Myocytes ◽  
Lactate And Pyruvate ◽  
Time Courses ◽  
Kinetics Of

1. Time courses for the uptake of L-lactate, D-lactate and pyruvate into isolated cardiac ventricular myocytes from guinea pig were determined at 11 degrees C or 0 degrees C (for pyruvate) in a citrate-based buffer by using a silicone-oil-filtration technique. These conditions enabled initial rates of transport to be measured without interference from metabolism of the substrates. 2. At a concentration of 0.5 mM, transport of all these substrates was inhibited by approx. 90% by 5 mM-alpha-cyano-4-hydroxycinnamate; at 10 mM-L-lactate a considerable portion of transport could not be inhibited. 3. Initial rates of L-lactate and pyruvate uptake in the presence of 5 mM-alpha-cyano-4-hydroxycinnamate were linearly related to the concentration of the monocarboxylate and probably represented diffusion of the free acid. The inhibitor-sensitive component of uptake obeyed Michaelis-Menten kinetics, with Km values for L-lactate and pyruvate of 2.3 and 0.066 mM respectively. 4. Pyruvate and D-lactate inhibited the transport of L-lactate, with Ki values (competitive) of 0.077 and 6.6 mM respectively; the Ki for pyruvate was very similar to its Km for transport. The Ki for alpha-cyano-4-hydroxycinnamate as a non-competitive inhibitor was 0.042 mM. 5. These results indicate that L-lactate, D-lactate and pyruvate share a common carrier in guinea-pig cardiac myocytes; the low stereoselectivity for L-lactate over D-lactate and the high affinity for pyruvate distinguish it from the carrier in erythrocytes and hepatocytes. The metabolic roles for this novel carrier in heart are discussed.

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