Evidence from two-protonic-state reactivity probe kinetics that chymopapain in fresh nonfruit latex ofCarica papaya consists of multiple forms of chymopapain A. The value of catalytic site characteristics in the identification, classification, and characterization of the papaya cysteine proteinases papain, the chymopapains, and papaya proteinase ?

1985 ◽  
Vol 4 (2) ◽  
pp. 103-127 ◽  
Author(s):  
Keith Brocklehurst ◽  
Raymond McKee ◽  
Erdjan Salih ◽  
Harry Smith
Keyword(s):  
Catalytic Site ◽  
Cysteine Proteinases ◽  
Multiple Forms ◽  
Site Characteristics

scholarly journals Differences in the chemical and catalytic characteristics of two crystallographically ‘identical’ enzyme catalytic sites. Characterization of actinidin and papain by a combination of pH-dependent substrate catalysis kinetics and reactivity probe studies targeted on the catalytic-site thiol group and its immediate microenvironment

1987 ◽  
Vol 247 (1) ◽  
pp. 181-193 ◽  
Author(s):  
E Salih ◽  
J P G Malthouse ◽  
D Kowlessur ◽  
M Jarvis ◽  
M O'Driscoll ◽  
...  
Keyword(s):  
Ph Dependence ◽  
Thiol Group ◽  
Ion Pair ◽  
Low Ph ◽  
Catalytic Site ◽  
Cysteine Proteinases ◽  
Acidic Media ◽  
Acid Media ◽  
Catalytic Sites

The characteristics of actinidin (EC 3.4.22.14) and papain (EC 3.4.22.2), two cysteine proteinases whose catalytic-site regions appear to superimpose to a degree that approaches atomic co-ordinate accuracy of both crystal structures, were evaluated by determining (a) the pH-dependence in acid media of the acylation process of the catalytic act (k+2/Ks) using N alpha-benzoyl-L-arginine p-nitroanilide (L-Bz-Arg-Nan) as substrate and (b) the sensitivity of the reactivity of the catalytic-site thiol group and its pH-dependence to structural change in small, thiol-specific, two-protonic-state reactivity probes (2,2′-dipyridyl disulphide and methyl 2-pyridyl disulphide) where enzyme-probe contacts should be restricted to areas close to the catalytic site. Distortion of the catalytic sites of the two enzymes at pH less than 4 was evaluated over time-scales appropriate for both stopped-flow reactivity probe kinetics (less than or equal to 1-2 s) and steady-state substrate catalysis kinetics (3-5 min) by using the 2,2′-dipyridyl disulphide monocation as a titrant for non-distorted catalytic sites. This permitted a lower pH limit to be defined for valid kinetic analysis of both types. The behaviour of the enzymes at pH less than 4 requires a kinetic model in which the apparently biomolecular reaction of enzyme with probe reagent is separated from the process leading to loss of conformational integrity by a potentially reversible step. The acylation of actinidin with L-Bz-Arg-Nan in acidic media occurs in two protonic states, one produced by raising the pH across pKa less than 4 which probably characterizes the formation of -S-/-ImH+ ion pair (pKa approx. 3) and the other, of higher reactivity, produced by raising the pH across pKa 5.5, which may characterize rearrangement of catalytic-site geometry. The pH-dependence of the acylation of papain by L-Bz-Arg-Nan is quite different and is not influenced by protonic dissociation with pKa values in the range 5-6. The earlier conclusion that the acylation of papain depends on two protonic dissociations each with pKa approx. 4 was confirmed. This argument is now more firmly based because titration with 2,2′-dipyridyl disulphide permits the loss of conformational integrity to be taken into account in the analysis of the kinetic data at very low pH. Methyl 2-pyridyl disulphide was synthesized by reaction of pyridine-2-thione with methyl methanethiolsulphonate and its pKa at I = 0.1 was determined by spectral analysis at 307 nm to be 2.8.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of binding site-catalytic site signalling in cysteine proteinases by using substrate-derived two-protonic-state reactivity probes

1986 ◽  
Vol 14 (6) ◽  
pp. 1225-1226 ◽  
Author(s):  
KEITH BROCKLEHURST ◽  
DEVANAND KOWLESSUR ◽  
GEETA PATEL ◽  
WILLIAM TEMPLETON ◽  
EMRYS THOMAS ◽  
...  
Keyword(s):  
Binding Site ◽  
Catalytic Site ◽  
Cysteine Proteinases

Isolation and characterization of multiple forms of renin from bull kidney

1980 ◽  
Vol 623 (2) ◽  
pp. 317-328 ◽  
Author(s):  
Giuseppina Conio ◽  
Pietro Ghiani ◽  
Eligio Patrone ◽  
Vincenzo Trefiletti ◽  
Biancamaria Uva ◽  
...  
Keyword(s):  
Multiple Forms ◽  
Isolation And Characterization

Mitochondrial aspartate transaminase. II. Isolation and characterization of the multiple forms

Biochemistry ◽  
1969 ◽  
Vol 8 (3) ◽  
pp. 1095-1105 ◽  
Author(s):  
Catherine M. Michuda ◽  
Marino Martinez-Carrion
Keyword(s):  
Aspartate Transaminase ◽  
Multiple Forms ◽  
Isolation And Characterization

scholarly journals Isolation and Characterization of Multiple Forms of Glutamate-Aspartate Aminotransferase from Pig Heart

1967 ◽  
Vol 242 (10) ◽  
pp. 2397-2409 ◽  
Author(s):  
M. Martinez-Carrion ◽  
C. Turano ◽  
E. Chiancone ◽  
F. Bossa ◽  
A. Giartosio ◽  
...  
Keyword(s):  
Aspartate Aminotransferase ◽  
Multiple Forms ◽  
Isolation And Characterization
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