Labeling of amide linkages in active site mapping: Carbonium ion and extended photoaffinity labeling approaches

Abstract

Communications to the Editor 7421 rium from Scheme I with an equation similar to eq 1 ;5b using the known values for K2K2 and K4 we may estimate K2 and K2 to be < 10"11 and > 1, respectively. The ionization pAla for the amidine {K2) is relatively low presumably owing to the powerfully electron-withdrawing ammonio group.Assuming equilibrium of the species II-VI and rate-limiting proton-catalyzed hydrolysis of the free carbodiimide VI, we may estimate the acid plateau rate constant (pH 0-4) from proton attack on the trimethylammonio model (&h = 3 X 102 M-1 s_1). The overall rate constant for reagent I becomes kt\K\K2K2/Kh5q in the acid plateau region which gives 3 X 10-2 s_l on substituting values for the equilibrium constants and & • The estimated value is much larger than that observed (10~3 s_1) indicating that one of the steps ]is rate limiting for the carbodiimide mechanism. The decomposition rate constant for VI increases with decrease in pH and it is likely that K2 becomes the rate-limiting step because this is pH in-

Alkylation of amide linkages and cleavage of the C chain in the enzyme-activated-substrate inhibition of .alpha.-chymotrypsin with N-nitrosamides

“…Thus, the minimum ratio of stable to labile benzyl groups arising from the alkylation of chymotrypsin was about 3:1. This result is considerably different from such ratios obtained in model studies; for example, the reaction of deaminatively formed benzyl carbonium ions with /V-ethylacetamide led to an Nbenzyl/O-benzyl ratio of 1 /6 (White et al, 1978;J. Cousins, unpublished work). Either the active site is constraining the reacting molecules in some way, or the inhibition involves benzyl diazonium ions rather than benzyl carbonium ions in the relatively hydrophobic active site.…”

“…The lie enzyme a-chymotrypsin is rapidly and irreversibly inhibited by the active-site-directed enzyme-activated substrates D-A-nitroso-A-benzyl-A-isobutyrylalaninamide (1), d-A- nitroso-A-benzyl-A-isobutyrylphenylalaninamide [2; asterisk (*) = '2c, 13C, or 14C], and A-nitroso-l,4-dihydro-6,7-dimethoxy-3(2A)-isoquinolinone (3) (White et al, 1975(White et al, , 1977(White et al, , 1978(White et al, , 1981. Inhibition results when the enzyme is alkylated by the benzyl carbonium ions produced (eq 1); fully inhibited a-chymotrypsin 2isobutyryl-Phe-O-Ser-Enz + a-chymotrypsin c6h5ch2n=n-o---C6H5CH2+-• benzylchymotrypsin (1) a-chymotrypsin has a benzyl/enzyme ratio of approximately 1 (White et al, 1981).…”

“…to inhibition thus has promise as a method that will map the active site of a-chymotrypsin with respect to the site of release of the carbonium ion. No other method of inhibition of enzymes (Sinnott, 1982; Silverman & Hoffman, 1984) involves intermediates powerful enough to react directly with amide linkages, with the exception of photoaffinity labelling (White et al, 1978;Bayley & Knowles, 1977). The amide linkages of proteins have been previously utilized in chemical reactions but only through intramolecular interactions (Glazer, 1976).…”

Membrane protein topology: amino acid residues in a putative transmembrane .alpha.-helix of bacteriorhodopsin labeled with the hydrophobic carbene-generating reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine

“…On the other hand, additional sites for peptide cleavage may be generated upon reaction of the carbene with the polypeptide backbone. These reactions are presumed to yield imido ester (White et al, 1978) which are highly susceptible to acid hydrolysis.…”

Fluorogenic N-Nitrosoamides: Active-Site Labeling Reagents for Chymotrypsin-like Proteases

“…IV-Nitrosoamide derivatives of amino acids are activesite-directed enzyme-activated inhibitors ("suicide inhibitors") for hydrolytic enzymes (White et al, 1975;1977a;1977b;1981;1990;Donadío et al, 1985; White and Chen, 1993). Because of the extremely high reactivity of the carbocations (White et al, 1968;1973;1978) released during enzyme-catalyzed hydrolyses, they can serve as relatively undiscriminating active-site labeling reagents (eq 1, Scheme 1). The alkylation by the carbocations can occur at the amide linkages as well as the side chains (except alkyl groups) within the active site of a targeted enzyme (Donadío et al, 1985; White et al, 1990).…”