Multiple alkylation and mapping of the active site of .alpha.-chymotrypsin by carbonium ions generated with active-site-directed enzyme-activated nitrosoamide substrates

White, Emil H.; Li, Min; Cousins, Joseph P.; Roswell, David F.

doi:10.1021/ja00161a048

Cited by 12 publications

(8 citation statements)

References 5 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Therefore the linkage between the label and the peptide must be labile towards the strong acid hydrolysis required for amino acid analysis. The labelled site was initially thought to be either on the side-chain oxygen atom of Ser3(cx) or that of Asp6(a), since it was known that similar ether or ester type linkages decomposed under the same hydrolysis conditions [35].…”

Section: Discussionmentioning

confidence: 99%

Photoaffinity labelling of cyanomethaemoglobin with derivatives of tryptophan and 5-bromotryptophan

Lin

Johnson

1995

Biochemical Journal

View full text Add to dashboard Cite

Tryptophan and 5-bromotryptophan (5-BrTrp) are relatively potent inhibitors of sickle-haemoglobin polymerization. The binding sites of these compounds to normal and sickle haemoglobin (HBA and HBS) have been suggested, but not firmly established, through the use of spin-labelled derivatives and/or computer modeling. In the present study we approached the problem by utilizing the technique of photoaffinity labelling. The cyanomet forms of HBA and HBS were subjected to photoaffinity labelling with N alpha-(4-azidotetrafluorobenzoyl)tryptophan and N alpha-(1-ethyl-2-diazomalonyl)-5-bromotryptophan respectively. Both irradiated samples of HBA and HBS were denatured, digested with trypsin, and then separated by reversed-phase HPLC. A labelled tryptic peptide was isolated from the photolabelling of HBS with N alpha-(1-ethyl-2-diazomalonyl)-5-bromotryptophan. The peptide was identified to be Val1(alpha)-Lys7(alpha), with the label attached to Val1(alpha), by virtue of amino acid analysis and sequencing, in conjunction with fast-atom-bombardment MS. The binding mode of N alpha-(1-ethyl-2-diazomalonyl)-5-bromotryptophan is proposed and its relevance to the potency of the 5-BrTrp-based anti-sickling agents is discussed.

show abstract

Section: Discussionmentioning

confidence: 99%

Photoaffinity labelling of cyanomethaemoglobin with derivatives of tryptophan and 5-bromotryptophan

Lin

Johnson

1995

Biochemical Journal

View full text Add to dashboard Cite

show abstract

“…Abboderin and Fruton's (1966) work suggested the participation of two carboxyl groups in the enzymatic action, while later it was shown (Carraway et al, 1969) that one of those residues was From the X-ray structure of pancreatic trypsin inhibitor-trypsin complex (Huber et al, 1974a), it can be seen that the reactive diazonium ion moiety is far from Asp-102, but fairly close to Asp-194 (Figure 4). Other nucleophiles that are within atom contact distance are From the 13C NMR studies, the possibility that alkylation occurs at the nitrogen atoms of Gin-192 or His-57 can be ruled out because no 13C peaks in the region of 50-60 ppm (the -CH2-N < region) were observed (White et al, 1990) (/V-alkyl derivative are stable to hydrolysis). S-Alkylation of 220 followed by cleavage to give S-alkyl cysteines is also ruled out based on the same rationale; that is, -CH2-Sis expected to give a peak near 36 ppm (White et al, 1990).…”

Section: Discussionmentioning

confidence: 99%

“…(5) 13C NMR of 6 N HCl Hydrolysates of [I-l3C] lcand D2a-Inhibited Trypsin. [1-13C] lc-inhibited trypsin (12 mg) after G-25 chromatography was hydrolyzed (White et al, 1990) [native trypsin (12 mg) as a control was treated in the same way]. The residue from the hydrolysis was dissolved in 0.60 mL of 0.1 N DC1 in D20 containing 1.7% CH3CN.…”

Section: Methodsmentioning

confidence: 99%

“…The D2a-inhibited enzyme solution (17 mg) (from part 4 above), after incubation with 0.09 M hydroxylamine at pH 7.2 for 6 h, was lyophilized. The residue was hydrolyzed (White et al, 1990), and the hydrolysates were dissolved in 0.50 mL of 0.1 N DC1 in D20; 13C NMR spectra were obtained (Figure 2).…”

Section: Methodsmentioning

confidence: 99%

“…followed by acidification to pH 2.2. The 13C NMR spectrum showed that two-thirds of the 67.2 ppm peak disappeared; the remaining third is also present in the spectrum of native trypsin and is attributed to the beta-carbons of the 10 threonine residues of the enzyme (White et al, 1990). A peak at 62.9 ppm grew in intensity; this peak has the same chemical shift (62.9 ppm) as that of [ 1 -l3C] 4-amino-1butanol measured under the same conditions.…”

Section: N=0mentioning

confidence: 99%

See 2 more Smart Citations

Inhibition of Trypsin with Active-Site-Directed Enzyme-Activated N-Nitrosoamide Substrates

White

Chen²

1995

Biochemistry

Self Cite

View full text Add to dashboard Cite

A series of active-site-directed enzyme-activated nitrosoamide inhibitors of trypsin has been designed, synthesized, and tested. The inhibitors contain an N-nitrosoamide group that can generate an alkylating agent and a positively charged ammonium ion group at the end of an aliphatic carbon chain that provides specificity. The half-lives of inhibition under normal conditions were 0.6 to 2 min for compounds in series 1. One of the compounds, N-(4-amino-1-butyl)-N-nitrosobenzamide (1b), is a very efficient inhibitor; its partition ratio, k2/kinact, is zero suggesting that it may be a useful titrant for trypsin and related enzymes. The extent of inhibition is substantially decreased by the competitive inhibitor benzamidine, indicating that the inhibitors were operating in the active site. Two modes of inhibition were noted: reversible and irreversible. The N-nitrosoamide inhibitors bind to the trypsin binding pocket guided by the primary specificity. They then acylate the enzyme (at Ser-195), producing a leaving group that generates diazonium ions (or) carbocations in the active site; these react with a proximal carboxylic acid side chain of the enzyme to form a carboxylic acid ester, presumably that of Asp-194. If primary amino groups are present on the alkyl group, the ester (13C NMR delta 67.2 ppm for R1COO13CH2-(CH2)nNH2) rearranges into the amide form (13C NMR delta 62.9 ppm for R1CONH(CH2)n13CH2OH) through an O-->N acyl migration; an irreversibly inhibited enzyme results. A model based on the orientation of the site-specific group of N-(4-amino-1-butyl)-N-nitroso-N'-isobutyryl-D-alaninamide (D2a) and its L antipode in the active site of trypsin is proposed to explain the preferential inhibition of trypsin by the D-isomer. Analysis of the structure--inhibition relationship revealed four factors that determine the inhibition modes of the inhibitors: 1, length of the alkyl group; 2, stability of the acyl-enzyme; 3, the option O--N acyl migration; and 4, chirality.

show abstract

The synthesis of N‐[4‐dimethylamino‐1‐butyl(1‐¹³C)‐N‐nitrosobenzamide], an inhibitor of tryspin; Cyanide does not exchange with acetonitrile

White

Chen

1993

Labelled Comp Radiopharmac

View full text Add to dashboard Cite

SUMMARYThe synthesis of N-[4-dimethylamino-l-butyl( l-13C)-N-nitrosobenzamide, a powerful active-site-directed inhibitor of trypsin, is reported. An efficient use of '3C-cyanide ion was achieved in the reaction with l-chloro-3-dimethylaminopropane, the first step of the synthesis. Acetonitrile was used as a co-solvent with water in that step; no evidence was found for cyanide ion exchange with acetonitrile. The earlier report of such an exchange appears to be in error; the formation of hydrocyanic acid under the reaction conditions can account for the observations.

show abstract

Multiple alkylation and mapping of the active site of .alpha.-chymotrypsin by carbonium ions generated with active-site-directed enzyme-activated nitrosoamide substrates

Cited by 12 publications

References 5 publications

Photoaffinity labelling of cyanomethaemoglobin with derivatives of tryptophan and 5-bromotryptophan

Photoaffinity labelling of cyanomethaemoglobin with derivatives of tryptophan and 5-bromotryptophan

Inhibition of Trypsin with Active-Site-Directed Enzyme-Activated N-Nitrosoamide Substrates

The synthesis of N‐[4‐dimethylamino‐1‐butyl(1‐¹³C)‐N‐nitrosobenzamide], an inhibitor of tryspin; Cyanide does not exchange with acetonitrile

Contact Info

Product

Resources

About

Multiple alkylation and mapping of the active site of .alpha.-chymotrypsin by carbonium ions generated with active-site-directed enzyme-activated nitrosoamide substrates

Cited by 12 publications

References 5 publications

Photoaffinity labelling of cyanomethaemoglobin with derivatives of tryptophan and 5-bromotryptophan

Photoaffinity labelling of cyanomethaemoglobin with derivatives of tryptophan and 5-bromotryptophan

Inhibition of Trypsin with Active-Site-Directed Enzyme-Activated N-Nitrosoamide Substrates

The synthesis of N‐[4‐dimethylamino‐1‐butyl(1‐13C)‐N‐nitrosobenzamide], an inhibitor of tryspin; Cyanide does not exchange with acetonitrile

Contact Info

Product

Resources

About

The synthesis of N‐[4‐dimethylamino‐1‐butyl(1‐¹³C)‐N‐nitrosobenzamide], an inhibitor of tryspin; Cyanide does not exchange with acetonitrile