Reaction of the active site of papain with a reporter group labeled phenacyl halide

1970 ◽  
Vol 92 (23) ◽  
pp. 6980-6982 ◽  
Author(s):  
Emil T. Kaiser ◽  
Richard W. Furlanetto
Keyword(s):  
Active Site ◽  
Reporter Group

Environment of a reporter group at the active site of chymotrypsin

1967 ◽  
Vol 89 (23) ◽  
pp. 5945-5951 ◽  
Author(s):  
Merrill B. Hille ◽  
Daniel E. Koshland
Keyword(s):  
Active Site ◽  
Reporter Group

scholarly journals Preferential nitration with tetranitromethane of a specific tyrosine residue in penicillinase from Staphylococcus aureus PCl. Evidence that the preferentially nitrated residue is not part of the active site but that loss of activity is due to intermolecular cross-linking

1978 ◽  
Vol 169 (2) ◽  
pp. 381-388 ◽  
Author(s):  
A F Bristow ◽  
R Virden
Keyword(s):  
Catalytic Activity ◽  
Active Site ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Normal Activity ◽  
Enzymic Activity ◽  
Cross Linking ◽  
Partial Loss ◽  
Tyrosine Residues ◽  
Reporter Group

1. Nitration of tyrosine residues of staphylococal penicillinase was accompanied by a partial loss of enzymic activity, which was not readily explained by nitration of a single residue. 2. Loss of activity correlated with low recovery of tyrosine plus nitrotyrosine, which was consistent with cross-linking. 3. The fraction of treated enzyme that was eluted from Sephadex G-75 earlier than native penicillinase was similar to the fraction of enzyme activity lost. Protein eluted in positions corresponding to monomer, dimer and higher oligomers respectively showed major bands in corresponding positions in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, indicating that the increase in molecular weight was due to intermolecular cross-linking. Monomeric enzyme containing up to 4 mol of nitrotyrosine/mol retained full catalytic activity. Dimeric enzyme retained 50% of normal activity, whereas higher oligomers retained an average of 8-15% of normal activity. 4. Monomeric enzyme isolated after treatment with equimolar tetranitromethane was nitrated predominantly at tyrosine-72.5. Reaction of reduced nitrated monomer with 1,5-difluoro-2,4-dinitrobenzene gave a monomeric, apparently cross-linked product with full catalytic activity. 6. It is concluded that tyrosine-72 plays no part in the active site. Its preferential nitration may be due to its being insufficiently exposed to be available for intermolecular cross-linking. This poperty may make it useful for attachment of a reporter group.

A Novel Restricted Photoaffinity Spin-Labeled Non-Nucleoside ATP Analogue as a Covalently Attached Reporter Group of the Active Site of Myosin Subfragment 1†

Biochemistry ◽  
2002 ◽  
Vol 41 (8) ◽  
pp. 2609-2620 ◽  
Author(s):  
Xiaoru Chen ◽  
Jean Grammer ◽  
J. David Lawson ◽  
Roger Cooke ◽  
Edward Pate ◽  
...  
Keyword(s):  
Active Site ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1 ◽  
Reporter Group

Ionization of a nitrophenol-containing reporter group at the active site of papain

Biochemistry ◽  
1974 ◽  
Vol 13 (4) ◽  
pp. 690-698 ◽  
Author(s):  
S. D. Lewis ◽  
J. A. Shafer
Keyword(s):  
Active Site ◽  
Reporter Group

scholarly journals Probing the active site of cytoplasmic aldehyde dehydrogenase with a chromophoric reporter group

1994 ◽  
Vol 300 (1) ◽  
pp. 25-30 ◽  
Author(s):  
T M Kitson ◽  
K E Kitson
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Active Site ◽  
Negative Charge ◽  
Positive Charge ◽  
Gel Filtration ◽  
Modified Enzyme ◽  
Covalently Linked ◽  
Reporter Group

3,4-Dihydro-3-methyl-6-nitro-2H-1,3-benzoxazin-2-one (‘DMNB’) reacts with cytoplasmic aldehyde dehydrogenase in a similar way to that previously observed with the structurally related p-nitrophenyl dimethylcarbamate, but provides a covalently linked p-nitrophenol-containing reporter group at the enzyme's active site. The pKa of the enzyme-linked reporter group is much higher than that of free p-nitrophenol, which is consistent with its being in a very hydrophobic environment, or possibly one containing negative charge. Upon binding of NAD+ to the modified enzyme, the pKa falls dramatically, by about 4 1/2 pH units. This implies that under these conditions there is a positive charge near the p-nitrophenoxide moiety, perhaps that of the nicotinamide ring of NAD+. The modified enzyme binds NAD+ very tightly; neither gel filtration nor dialysis is effective in separating them. However, the reporter group provides a convenient way of monitoring the displacement of this bound NAD+ when NADH is added.

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