Mechanism of the reaction of papain with substrate-derived diazomethyl ketones Implications for the difference in site specificity of halomethyl ketones for serine proteinases and cysteine proteinases and for stereoelectronic requirements in the papain catalytic mechanism

1979 ◽  
Vol 177 (3) ◽  
pp. 769.b1-769.b1
Keyword(s):  
Catalytic Mechanism ◽  
Site Specificity ◽  
Serine Proteinases ◽  
Cysteine Proteinases ◽  
The Difference ◽  
Diazomethyl Ketones ◽  
Mechanism Of The Reaction

scholarly journals Mechanism of the reaction of papain with substrate-derived diazomethyl ketones. Implications for the difference in site specificity of halomethyl ketones for serine proteinases and cysteine proteinases and for stereoelectronic requirements in the papain catalytic mechanism

1978 ◽  
Vol 175 (2) ◽  
pp. 761-764 ◽  
Author(s):  
K Brocklehurst ◽  
J P G Malthouse
Keyword(s):  
Catalytic Mechanism ◽  
Formation Rate ◽  
Thiol Group ◽  
Site Specificity ◽  
Serine Proteinases ◽  
Cysteine Proteinases ◽  
Cyclic Transition ◽  
Cyclic Transition State ◽  
The Difference ◽  
Diazomethyl Ketones

The reactions of papain (EC 3.4.22.2) with substrate-derived diazomethyl ketones reported by Leary, Larsen, Watanabe & Shaw [Biochemistry (1977) 16, 5857–5861] are unusual in that (i) these reagents fail to react with low-molecular-weight thiols and (ii) the rate of reaction with the papain thiol group does not decrease to near-zero values across a pKa of 4 as the pH is decreased. Existing data are shown to suggest an interpretation involving neighbouring-group participation via transient thiohemiketal formation, rate-determining protonation by imidazolium ion and alkylation on sulphur via a three-membered cyclic transition state. Implications for (a) the difference in site-specificity exhibited by halomethyl ketones in their reactions with serine proteinases and cysteine proteinases and (b) stereoelectronic requirements in the mechanism of papain-catalysed hydrolysis are discussed. The possibility of two tetrahedral intermediates between adsorptive complex and acyl-enzyme is indicated.

scholarly journals The synthesis of peptidylfluoromethanes and their properties as inhibitors of serine proteinases and cysteine proteinases

1986 ◽  
Vol 239 (3) ◽  
pp. 633-640 ◽  
Author(s):  
P Rauber ◽  
H Angliker ◽  
B Walker ◽  
E Shaw
Keyword(s):  
Cathepsin B ◽  
Cysteine Proteinase ◽  
Bond Formation ◽  
Serine Proteinase ◽  
Covalent Bond ◽  
Serine Proteinases ◽  
Cysteine Proteinases ◽  
Active Centre ◽  
Covalent Bond Formation ◽  
The Difference

A synthesis of peptidylfluoromethanes is described that utilizes the conversion of phthaloyl amino acids into their fluoromethane derivatives. These can be deblocked and elongated. The inactivation of chymotrypsin by Cbz-Phe-CH2F (benzyloxycarbonylphenylalanylfluoromethane) was found to be considerably slower than that of the analogous chloromethane. The fluoromethane analogue inactivates chymotrypsin with an overall rate constant that is 2% of that observed for the inactivation of the enzyme with the chloromethane. However, the result is the same. The reagent complexes in a substrate-like manner, with Ki = 1.4 × 10(-4) M, and alkylates the active-centre histidine residue. Cbz-Phe-Phe-CH2F and Cbz-Phe-Ala-CH2F were investigated as inactivators of the cysteine proteinase cathepsin B. The difference in reactivity between fluoromethyl ketones and chloromethyl ketones is less pronounced in the case of the cysteine proteinase than for the serine proteinase. Covalent bond formation takes place in this case also, as demonstrated by the use of a radiolabelled reagent.

The structure and mechanism of action of papain

1970 ◽  
Vol 257 (813) ◽  
pp. 237-248 ◽  
Keyword(s):  
Mechanism Of Action ◽  
Catalytic Mechanism ◽  
Proteolytic Enzymes ◽  
Cysteine Proteinase ◽  
Preceding Paper ◽  
Cysteine Residue ◽  
Enzymic Activity ◽  
Critical Survey ◽  
Cysteine Proteinases ◽  
Stem Bromelain

The cysteine proteinases form a group of enzymes which depend for their enzymic activity on the thiol group of a cysteine residue. Several which occur in plants have been investigated extensively and include papain, ficin and stem bromelain (Smith & Kimmel i960). Although the term papain, introduced last century to describe the proteolytic principle in papaya latex (Wurtz & Bouchut 1879) is still used to describe crude dried latex, the crystalline enzyme is readily obtained (Kimmel & Smith 1954). Ficin is known to consist of several closely related enzymes which have been resolved (Sgarbieri, Gupte, Kramer & Whitaker 1964), but for most structural and mechanistic studies the unresolved mixture of enzymes has been used. Stem bromelain also appears to be a mixture of at least two proteolytic enzymes which have not yet been resolved (Ota, Moore & Stein 1962; Murachi 1964). In spite of the recognized heterogeneity of ficin and stem bromelain, it does seem that both structurally and mechanistically they are similar to papain. Only one bacterial cysteine proteinase has received a detailed study, namely, streptococcal proteinase, and it appears to have little or no relation in its amino acid sequence with the plant enzymes (Liu, Stein, Moore & Elliott 1965). The functional groups involved in the catalytic mechanism are apparently the same as in the plant proteinases (Gerwin, Stein & Moore 1966; Liu 1967; Husain & Lowe 1968 a , c ), but the mechanism of action has not been extensively studied. It may well be however that the plant and bacterial cysteine proteinases have converged onto a similar mechanism of action by two independent evolutionary pathways, as now seems apparent for the animal and bacterial serine proteinases (Alden, Wright & Kraut, this volume, p. 119). Because the tertiary crystal structure of papain (Drenth, Jansonius, Koekoek, Swen & Wolthers 1968; see also the preceding paper, p. 231) is now known, a critical survey of this enzyme is apposite.

Picornaviral 3C cysteine proteinases have a fold similar to chymotrypsin-like serine proteinases

Nature ◽  
1994 ◽  
Vol 369 (6475) ◽  
pp. 72-76 ◽  
Author(s):  
Marc Allaire ◽  
Maia M. Chernaia ◽  
Bruce A. Malcolm ◽  
Michael N. G. James
Keyword(s):  
Serine Proteinases ◽  
Cysteine Proteinases

scholarly journals Synthesis and properties of peptidyl derivatives of arginylfluoromethanes

1988 ◽  
Vol 256 (2) ◽  
pp. 481-486 ◽  
Author(s):  
H Angliker ◽  
P Wikström ◽  
P Rauber ◽  
S Stone ◽  
E Shaw
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Active Site ◽  
Relative Effectiveness ◽  
Serine Proteinases ◽  
Cysteine Proteinases ◽  
Peptide Derivatives ◽  
Guanidino Group ◽  
Derivatives Of

Two peptide derivatives of arginylfluoromethane (Arg-CH2F), namely Bz(benzoyl)-Phe-ArgCH2F and D-Phe-Pro-Arg-CH2F, have been synthesized by extension of available methods, i.e. the Dakin-West reaction [Rasnick (1985) Anal. Biochem. 149, 461-465] or synthesis of a phthaloyl-blocked C-terminal fluoromethane [Rauber, Angliker, Walker & Shaw (1986) Biochem. J. 239, 633-640; Angliker, Wikström, Rauber & Shaw (1987) Biochem. J. 241, 871-875] with subsequent elongation. The guanidino group of arginine was protected as the bis-Cbz (benzyloxycarbonyl) derivative. The products were examined as active-site-directed inhibitors of some trypsin-related serine proteinases as well as a pair of cysteine proteinases. The results extend previous observations that the rate of alkylation of serine proteinases by fluoromethanes may be considerably slower than by chloromethanes. As expected, the amino acid sequence of the inhibitors influenced their relative effectiveness. Thus the rate of inactivation of a number of trypsin-like proteinases by D-Phe-Pro-Arg-CH2F varied by more than two orders of magnitude.

Measurement of Elastase and Cysteine Proteinases in Synovial Fluid of Patients with Rheumatoid Arthritis, Sero-Negative Spondylarthropathies, and Osteoarthritis

1992 ◽  
Vol 38 (9) ◽  
pp. 1694-1697 ◽  
Author(s):  
G Huet ◽  
R M Flipo ◽  
C Richet ◽  
C Thiebaut ◽  
D Demeyer ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Synovial Fluid ◽  
Proteinase Inhibitor ◽  
Cysteine Proteinase ◽  
Cartilage Degradation ◽  
Enzyme Assays ◽  
Cysteine Proteinases ◽  
Inhibitor Complex ◽  
The Difference ◽  
Cysteine Proteinase Activity

Abstract Synovial fluid samples were collected from 45 patients with rheumatoid arthritis, spondylarthropathy, or osteoarthritis, to study their content of elastase (EC 3.4.21.37) and of cysteine proteinases (EC 3.4.22.1, 3.4.22.15). We measured both elastase complexed with alpha 1-proteinase inhibitor and elastase activity toward the substrate L-pyroglutamyl-L-prolyl-L-valine-p-nitroanilide. Cysteine proteinase activities were measured with the substrates N-benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-amido-4-methylcoumarin (Z-Phe-Arg-AMC) and Z-Arg-Arg-AMC and the inhibitor E-64 [L-trans-epoxysuccinyl-leucyl-amido-(4-guanidino)-butane]. In all these enzyme assays, higher median values were obtained in inflammatory arthropathies than in osteoarthritis. The concentration of the elastase-alpha 1-proteinase inhibitor complex and of elastase and cysteine proteinase activities were statistically higher in patients with rheumatoid arthritis than in patients with osteoarthritis. The difference in results between patients with spondylarthropathy and patients with osteoarthritis was statistically significant only for the elastase-alpha 1-proteinase inhibitor complex. The median values of the complex and of both enzyme activities were higher in patients with rheumatoid arthritis than in patients with spondylarthropathy; however, the difference was statistically significant only for the cysteine proteinase activity measured with Z-Arg-Arg-AMC substrate. These results suggest that both elastase and cysteine proteinases, which are increased in patients with inflammatory arthritis, are involved in cartilage degradation in these arthropathies.

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