scholarly journals Escherichia coli alkaline phosphatase. An analysis of transient kinetics

1971 ◽  
Vol 125 (1) ◽  
pp. 319-327 ◽  
Author(s):  
S. E. Halford
Keyword(s):  
Escherichia Coli ◽  
Alkaline Phosphatase ◽  
Inorganic Phosphate ◽  
Active Sites ◽  
Substrate Binding ◽  
Equilibrium Mixture ◽  
Stopped Flow ◽  
Transient Kinetics ◽  
Substrate Complex ◽  
Catalytically Active

1. The hydrolysis of 2,4-dinitrophenyl phosphate by Escherichia coli alkaline phosphatase at pH5.5 was studied by the stopped-flow technique. The rate of production of 2,4-dinitrophenol was measured both in reactions with substrate in excess of enzyme and in single turnovers with excess of enzyme over substrate. It was found that the step that determined the rate of the transient phase of this reaction was an isomerization of the enzyme occurring before substrate binding. 2. No difference was observed between the reaction after mixing a pre-equilibrium mixture of alkaline phosphatase and inorganic phosphate, with 2,4-dinitrophenyl phosphate at pH5.5 in the stopped-flow apparatus, and the control reaction in which inorganic phosphate was pre-equilibrated with the substrate. Since dephosphorylation is the rate-limiting step of the complete turnover at pH5.5, this observation suggests that alkaline phosphatase can bind two different ligands simultaneously, one at each of the active sites on the dimeric enzyme, even though only one site is catalytically active at any given time. 3. Kinetic methods are outlined for the distinction between two pathways of substrate binding, which include an isomerization either of the free enzyme or of the enzyme–substrate complex.

ELECTRON MICROSCOPY OF ALKALINE PHOSPHATASE OF ESCHERICHIA COLI

1966 ◽  
Vol 12 (4) ◽  
pp. 605-607 ◽  
Author(s):  
V. M. Kushnarev ◽  
T. A. Smirnova
Keyword(s):  
Electron Microscopy ◽  
Escherichia Coli ◽  
Alkaline Phosphatase ◽  
Cell Wall ◽  
Enzyme Activity ◽  
Electron Microscope ◽  
Inorganic Phosphate ◽  
E Coli

A method is described for determining the localization of alkaline phosphatase in the cells of E. coli B with the electron microscope. Enzyme activity, determined by deposition of inorganic phosphate, is located in the exterior layer of the cell wall.

scholarly journals Studies on alkaline phosphatase. Phosphorylation of calf intestinal alkaline phosphatase by 32P-labelled pyrophosphate

1968 ◽  
Vol 107 (2) ◽  
pp. 279-283 ◽  
Author(s):  
H N Fernley ◽  
Sylvia Bisaz
Keyword(s):  
Alkaline Phosphatase ◽  
Intestinal Mucosa ◽  
Active Sites ◽  
Stopped Flow ◽  
Serine Residue ◽  
Intestinal Alkaline Phosphatase ◽  
Calf Intestinal Alkaline Phosphatase ◽  
Substrate Hydrolysis

1. A purified preparation of alkaline phosphatase from calf-intestinal mucosa was phosphorylated by 32P-labelled PPi at a serine residue on the enzyme. Under the conditions employed, up to 0·15μm-labelled sites were obtained from 1μm-[32P]PPi. 2. The phosphorylated enzyme was labile, the rate of dephosphorylation being similar to the overall rate of substrate hydrolysis. 3. A stopped-flow technique was used to determine the number of phosphomonoesterase active sites, which agreed with the number of 32P-labelled sites. 4. It is concluded that calf-intestinal alkaline phosphatase is both a phosphomonoesterase and a pyrophosphatase.

scholarly journals Escherichia coli alkaline phosphatase. Relaxation spectra of ligand binding

1972 ◽  
Vol 126 (3) ◽  
pp. 727-738 ◽  
Author(s):  
S. E. Halford
Keyword(s):  
Escherichia Coli ◽  
Alkaline Phosphatase ◽  
Ligand Binding ◽  
Catalytic Mechanism ◽  
Temperature Jump ◽  
Relaxation Spectrum ◽  
Equilibrium Mixture ◽  
Difference Spectroscopy ◽  
Relaxation Spectra

The temperature-jump technique was used to study the binding equilibrium between the Escherichia coli alkaline phosphatase dimer and 2-hydroxy-5-nitrobenzyl phosphonate in 0.1m-tris buffer, pH8.0. Three partially discrete relaxations were observed, two of which could be related to the bimolecular associations of ligand with different conformations of the enzyme and the third to the interconversion of these states. Relaxation spectra were also used to analyse the changes in the mechanism of ligand binding to alkaline phosphatase caused by increase in ionic strength. The relaxation spectrum observed after the addition of Pi to the equilibrium mixture of phosphonate and enzyme was also studied. Difference spectroscopy indicated that both of these ligands were bound to the alkaline phosphatase dimer at the same time. These results are related to the catalytic mechanism of this enzyme, with particular reference to the role of two identical subunits in a dimeric enzyme that exhibits only one active site functioning in catalysis at any given time.

scholarly journals Expression of precursor and mature carnitine palmitoyltransferase II in Escherichia coli and in vitro: differential behaviour of rat and human isoforms

1993 ◽  
Vol 294 (1) ◽  
pp. 79-86 ◽  
Author(s):  
N F Brown ◽  
A Sen ◽  
D A Soltis ◽  
B Jones ◽  
D W Foster ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Active Sites ◽  
Expression System ◽  
Active Form ◽  
Carnitine Palmitoyltransferase ◽  
Coding Region ◽  
E Coli ◽  
Catalytically Active ◽  
Carnitine Palmitoyltransferase Ii

cDNAs corresponding to the precursor and mature forms of rat carnitine palmitoyltransferase II (CPT II) were found to be readily expressed in Escherichia coli. In both cases, catalytically active immunoreactive protein was produced and became largely membrane-associated. The precursor form of the enzyme was not proteolytically processed. Removal of 126 bp from the 5′ end of the cDNA coding region allowed expression of a truncated CPT II (lacking the N-terminal 17 residues of the mature protein), but this product was inactive. cDNAs encoding the precursor and mature forms of human CPT II resisted direct expression in E. coli. However, the impediment was overcome when the latter cDNA was ligated in-frame 3′ to sequence encoding a glutathione S-transferase. This construct yielded abundant quantities of the corresponding fusion protein, a portion of which was soluble and catalytically active. In vitro transcription and translation of the various cDNAs established that the lower mobility on SDS/PAGE of rat CPT II compared with its human counterpart (despite their identical numbers of amino acids) is an intrinsic property of the primary sequences of the proteins themselves. Also, the human cDNA was found to contain an artifactual termination signal for T3 RNA polymerase that could be bypassed by the T7 polymerase. Thus rat CPT II can be expressed in active form in E. coli with characteristics similar to those of the enzyme in mitochondria, opening the way to future location of active sites within the molecule. An alternative expression system will be needed for similar studies on human CPT II.

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