Current Status of the Structure of Papain: The Linear Sequence, Active Sulfhydryl Group, and the Disulfide Bridges

Light, Albert; Frater, R.; Kimmel, Joe R.; Smith, Emil L.

doi:10.1073/pnas.52.5.1276

Cited by 95 publications

(22 citation statements)

References 13 publications

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“…It is also possible that the methyl group may be attracted by hydrophobic interaction with the tryptophanyl residue which was shown to be adjacent to the active cysteine by Light et al [18]. In addition, the reactivity of the SH group may be enhanced by an interaction with the carboxylate group.…”

Section: A-amentioning

confidence: 99%

Stereospecific Alkylation with Asymmetric Reagents

Wallenfels

Eisele

1968

European Journal of Biochemistry

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1.When undergoing nucleophilic reactions with proteins, a-iodopropionic acid and its amide show greater specificity for SH groups than do iodoacetic acid and its amide.2. The kinetics and stereochemistry of the reactions of the D( +) and L( -) antipodes of a-iodopropionic acid and its amide with both cysteine and papain were investigated. The antipodes of the SH reagents reacted with these asymmetric SH compounds a t different rates. I n the reaction with papain, the L( -) antipode of the acid reacted faster than the D(+) antipode; the stereochemical preference was inverted when the amide was used, the D(+)antipode reacting faster than the L( -)antipode. 3.Arguing from the differing reactivities of the SH reagents and their antipodes and the pH dependence of the reaction rates, it has been suggested, that L( -)a-iodopropionic acid is specifically oriented onto the reaction center through the concerted attractive and repulsive actions of a cationic and an anionic group in the protein. These have been tentatively identified as a protonated imidazole residue and a carboxylate group respectively.4. Parallels between these stereospecific alkylations and the catalytic action of the enzyme are discussed.Gundlach, Stein, and Moore [1] demonstrated in their studies of the alkylation of ribonuclease with iodoacetic acid and iodoacetamide, that these "classic" sulfhydryl reagents do not react only with SH groups. Other nucleophiles present in proteins, such as amino, imidazolyl or thioether groups may also be alkylated. The extent of these side reactions depends on the reaction conditions. Our purpose in undertaking this work was to find SH reagents with higher selectivity for SH groups.Reasoning from a theory of Bunnett [a] it seemed likely, that this could be achieved by the introduction of polarizable substituents alpha to the halogen of halogen-alkyles. The possibility for the action of dispersion forces, which is introduced with the polarizable substituent, should specially favor the transition state for the reaction with the highly polarizable SH group. Steric, inductive or mesomeric effects of the a substituent would be expected to show the same influence on the reactions with all different nucleophilic groups.We tested this hypothesis using a series of derivatives of iodoacetamide with different substituents alpha to the iodine. With the methyl group as CI substituent i e . in the reaction of amino acids with a-iodopropionamide we observed an enhanced selectivity for SH groups as predicted by Bunnett's theory A further possibility for selective reactivity is contributed by a-substitution in iodoacetic acid derivatives ; the leaving group of the reagent is attached to an asymmetric carbon atom. One may expect specific interactions if the substrate itself is asymmetric since diastereomeric transition states are involved. This type of stereoselective reaction was shown for the first time by Heinrikson et al. [4,5]. They found, that the essential histidine (residue 12) of ribonuclease reacts preferentially with the D-...

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Section: A-amentioning

confidence: 99%

Stereospecific Alkylation with Asymmetric Reagents

Wallenfels

Eisele

1968

European Journal of Biochemistry

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“…Analysis of the sequence suggests that this gene is a papain-like cysteine protease. The predicted amino acid sequence of ALSCYP1 begins approximately 390 bp from the N terminus of other plant cysteine proteases and contains several of the elements characteristic of the structure of papain (Light et al, 1964;Mitchel et al, 1970). The three highly conserved amino acids (Cys, His and Asn) that make up the catalytic triad characteristic of the active site of cysteine proteases (Storer and Menard, 1994) are present.…”

Section: Isolation and Characterization Of A Partial Alstroemeria Cysmentioning

confidence: 99%

Cysteine protease gene expression and proteolytic activity during senescence of Alstroemeria petals

Wagstaff¹,

Leverentz²,

Griffiths³

et al. 2002

Journal of Experimental Botany

114

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The functional life of the flower is terminated by senescence and /or abscission. Multiple processes contribute to produce the visible signs of petal wilting and inrolling that typify senescence, but one of the most important is that of protein degradation and remobilization. This is mediated in many species through protein ubiquitination and the action of specific protease enzymes. This paper reports the changes in protein and protease activity during development and senescence of Alstroemeria flowers, a Liliaceous species that shows very little sensitivity to ethylene during senescence and which shows perianth abscission 8 -10 d after flower opening. Partial cDNAs of ubiquitin (ALSUQ1) and a putative cysteine protease (ALSCYP1) were cloned from Alstroemeria using degenerate PCR primers and the expression pattern of these genes was determined semi-quantitatively by RT-PCR. While the levels of ALSUQ1 only fluctuated slightly during floral development and senescence, there was a dramatic increase in the expression of ALSCYP1 indicating that this gene may encode an important enzyme for the proteolytic process in this species. Three papain class cysteine protease enzymes showing different patterns of activity during flower development were identified on zymograms, one of which showed a similar expression pattern to the cysteine protease cDNA.

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“…In the SHprotease from streptococci the presence of a histidine residue of pK = 6 has been also established [9,10] at the active site, yet in the group of plant SH-proteases (papin, ficin, bromelain) an ionizable group of pK = 3.9-4.3 was found. This finding seems to indicate the presence of a carboxyl group at the active site; in the case of papain an aspartic acid residue was suggested [3,11 ]. On the other hand, Husein and Lowe using a ditopic derivative of dibromoacetone were able to find a histidine molecule 5 A distant from the cys- value of the group ionizable in the acidic range.…”

Section: Resultsmentioning

confidence: 88%