Generation of Nucleophilic Character in the Cys25/His159 Ion Pair of Papain Involves Trp177 but Not Asp158

Gul, Sheraz; Hussain, Syeed; Thomas, Mark; Resmini, Marina; Verma, Chandra; Thomas, Emrys W.; Brocklehurst, Keith

doi:10.1021/bi702126p

Cited by 32 publications

(57 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The factors that govern the properties of the artificial, buried Glu-Lys pair are the same ones that govern the properties of ionizable groups in the active sites of enzymes (38)(39)(40). Therefore, the successful engineering of a Glu-Lys pair in the hydrophobic interior of a highly stable form of SNase is also significant because it constitutes an empirical demonstration that buried ion pairs and charge clusters resembling enzymatic active sites can be engineered in the hydrophobic interior of a scaffold protein.…”

Section: Discussionmentioning

confidence: 99%

Structural and thermodynamic consequences of burial of an artificial ion pair in the hydrophobic interior of a protein

Robinson

Castañeda

Schlessman

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

An artificial charge pair buried in the hydrophobic core of staphylococcal nuclease was engineered by making the V23E and L36K substitutions. Buried individually, Glu-23 and Lys-36 both titrate with pK a values near 7. When buried together their pK a values appear to be normal. The ionizable moieties of the buried Glu-Lys pair are 2.6 Å apart. The interaction between them at pH 7 is worth 5 kcal/mol. Despite this strong interaction, the buried Glu-Lys pair destabilizes the protein significantly because the apparent Coulomb interaction is sufficient to offset the dehydration of only one of the two buried charges. Save for minor reorganization of dipoles and water penetration consistent with the relatively high dielectric constant reported by the buried ion pair, there is no evidence that the presence of two charges in the hydrophobic interior of the protein induces any significant structural reorganization. The successful engineering of an artificial ion pair in a highly hydrophobic environment suggests that buried Glu-Lys pairs in dehydrated environments can be charged and that it is possible to engineer charge clusters that loosely resemble catalytic sites in a scaffold protein with high thermodynamic stability, without the need for specialized structural adaptations.electrostatics | internal charge | low-barrier hydrogen bond | dielectric constant | enzyme design P aired ionizable groups buried in hydrophobic environments inside proteins are an uncommon but essential structural motif necessary for H + transport (1), e − transfer (2), catalysis (3-5), and other important biochemical processes. Despite their importance, the properties of these buried ionizable pairs are poorly understood because they are difficult to study experimentally in the proteins where they play functional roles. When the pair consists of a basic and an acidic group it is usually assumed to be charged, but this is often just speculation without direct experimental support (6, 7). In principle, a Coulomb interaction between two ionizable groups of opposite polarity buried in a dehydrated environment inside a protein can be very strong, but this has not been established experimentally. Simulations suggest that buried ion pairs are always destabilizing (8); however, from an experimental perspective it is not known if the favorable Coulomb interaction would be sufficient to compensate for the unfavorable dehydration of the two buried charges or if the net effect of the pair on the thermodynamic stability of a protein would be stabilizing or not. These issues were examined in detail with an artificial ion pair buried in the hydrophobic interior of staphylococcal nuclease (SNase).The buried ion pair was engineered by introducing the V23E and L36K substitutions in a highly stable form of SNase. The pK a values of Glu-23 and Lys-36 introduced individually are anomalous (pK a of 7.1 for ref. 9; ref. 10), consistent with the groups existing in hydrophobic environments that are neither as polar nor as polarizable as water (11-13). Structural modeli...

show abstract

Section: Discussionmentioning

confidence: 99%

Structural and thermodynamic consequences of burial of an artificial ion pair in the hydrophobic interior of a protein

Robinson

Castañeda

Schlessman

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…The side chain of the latter assumes the role of the oxyanion hole of serine peptidases in stabilizing the tetrahedral intermediate (2) , whereas the former plays a role analogous to Asp in the catalytic triad of serine peptidases, forming a hydrogen bond to His159, but is not absolutely essential for catalysis (3) . Recent evidence, however, suggests an important role for the adjacent Trp177 in the generation of the nucleophilic character of the catalytic diad (4) .…”

Section: The Active Center Of Papain-like Peptidasesmentioning

confidence: 99%

Papain-like peptidases: structure, function, and evolution

Novinec

Lenarčič²

2013

BioMolecular Concepts

View full text Add to dashboard Cite

Papain-like cysteine peptidases are a diverse family of peptidases found in most known organisms. In eukaryotes, they are divided into multiple evolutionary groups, which can be clearly distinguished on the basis of the structural characteristics of the proenzymes. Most of them are endopeptidases; some, however, evolved into exopeptidases by obtaining additional structural elements that restrict the binding of substrate into the active site. In humans, papain-like peptidases, also called cysteine cathepsins, act both as non-specific hydrolases and as specific processing enzymes. They are involved in numerous physiological processes, such as antigen presentation, extracellular matrix remodeling, and hormone processing. Their activity is tightly regulated and dysregulation of one or more cysteine cathepsins can result in severe pathological conditions, such as cardiovascular diseases and cancer. Other organisms can utilize papainlike peptidases for different purposes and they are often part of host-pathogen interactions. Numerous parasites, such as Plasmodium and flukes, utilize papain-like peptidases for host invasion, whereas plants, in contrast, use these enzymes for host defense. This review presents a state-of-the-art description of the structure and phylogeny of papain-like peptidases as well as an overview of their physiological and pathological functions in humans and in other organisms.

show abstract

“…There is considerable variation among members of the papain family, however, in substrate specificity and catalytic-site reactivity [81,[153][154][155][156][157][158], which reveals that this view is correct only for low-resolution aspects of the mechanism, such as the roles of the cysteine and histidine components of the catalytic-site ion pair. The results of detailed mechanistic investigations on papain and some of its natural variants (discussed in [44,45]) have revealed that papain exhibits characteristics at one end of a spectrum of chemical behavior with actinidin at the other, and with caricain and ficin occupying intermediate positions. Key results include the identification of kinetically influential pK a values, both those associated with the catalytic site (Cys25)-S À /(His159)-Im þ H ion pair (papain numbering) and others more remote [44], the coupling of molecular recognition interactions with catalytic site chemistry, and postacylation protein dynamics.…”

Section: Cysteine Proteinasesmentioning

confidence: 99%

“…Studies using reactivity probes, particularly disulfides containing the 2-mercaptopyridine leaving group as two-protonic-state electrophiles [41,81], including substrate-derived probes [20,44], a 4-pyrimidyl probe that remains unprotonated over a wide range of low pH values [44] and 4-chloro-7-nitrobenzofurazan [159], pHdependent kinetics [42], computer modeling including normal mode analysis [45] and electrostatic potential calculations [81], and natural variants of the papain family, have changed the perception of the catalytic mechanisms of these enzymes and of the influence of binding interactions and electrostatic effects (see [20,44] for detailed discussion).…”

Section: Cysteine Proteinasesmentioning

confidence: 99%

“…The reactivity of a catalytic site nucleophile that plays a central role in the catalytic act is a prime target for such investigations, and studies on the cysteine proteinase papain and its natural variants [20] using specifically designed and synthesized thiol-specific probes demonstrated such dependence by large changes in pH-k profiles [44] as discussed in Section 11.3. Even if the inhibitors are not substrate analogs, experiments can be designed to use specific noncovalent interactions to reveal key aspects of the catalytic mechanism (e.g., [45]). …”

mentioning

confidence: 99%

See 1 more Smart Citation

Substrate Recognition

Brocklehurst

Gul

Pickersgill

2009

Amino Acids, Peptides and Proteins in Organic Chemistry

Self Cite

View full text Add to dashboard Cite

Specific molecular recognition processes by which molecules recognize and discriminate between closely related partners are involved in essentially all aspects of biological function, ranging in complexity from enzyme-inhibitor and enzymesubstrate systems to phenomena such as gene expression, the immune system, and synaptic transmission, and these are important considerations in drug design [1]. This chapter is concerned with general concepts exemplified by a selection of relatively well-defined enzyme-substrate and, by extension, enzyme-inhibitor systems. The original concept of recognition by complementarity in the enzymesubstrate adsorptive (Michaelis) complex was suggested by Emil Fischer in 1894 using his well-known lock-and-key analogy [2]. It is now generally accepted that this has now been superseded by complementarity in the reaction transition state, based on the ideas reported by J.B.S. Haldane [3] in 1930 and elaborated by Linus Pauling [4] in 1946. The catalytic potential of enzymes derives from stabilization in the transition state relative to any stabilization in the Michaelis complex. The latter is anticatalytic in that the free energy expended in stabilizing the adsorptive complex offsets the transition-state stabilization energy (e.g., [5]).Valuable chapters and books on enzyme catalysis and inhibition are available (e.g., [5][6][7][8][9][10][11]) and detailed mechanistic studies on many individual enzymes are described in Sinnot [12]. Macromolecular catalysis includes also catalysis by catalytic antibodies [13] and by synthetic polymers [14], including molecularly imprinted polymers [15]. The contributions of selections of various types of chemical catalysis such as acid-base, nucleophilic, and electrophilic catalysis [5,6,8] are almost always insufficient to account for the high catalytic efficiency of enzymes. A particularly important additional contribution is the entropic advantage that derives from the fact that enzyme catalysis occurs within an enzyme-substrate complex. The complex is essentially a single molecular species and the catalytic act involves one or more unimolecular steps during which there is no loss of translational or rotational entropy

show abstract

Generation of Nucleophilic Character in the Cys25/His159 Ion Pair of Papain Involves Trp177 but Not Asp158

Cited by 32 publications

References 27 publications

Structural and thermodynamic consequences of burial of an artificial ion pair in the hydrophobic interior of a protein

Structural and thermodynamic consequences of burial of an artificial ion pair in the hydrophobic interior of a protein

Papain-like peptidases: structure, function, and evolution

Substrate Recognition

Contact Info

Product

Resources

About