WHY CYSTEINE IS SPECIAL? SOME BASIC INFORMATION ABOUT CYSTEINE ITSELF Cysteine (Fig.

1) is one of 20 naturally occurring, 'biogenic' amino acids which linked by e tide bonds form oly e tides and roteins. !ike the other amino acids cysteine is abundant as !"form. #t is genetically encoded by two ossible codons (nucleotide tri lets of m$%&) '(' and '(C. )tructurally cysteine belongs to the sulfur amino acids, because of sulfur atom a earing in its side chain. *he sulfur atom of cysteine is in+ol+ed in formation the sulfhydryl grou which is +ery reacti+e. *hat differs cysteine from another sulfur amino acid " methionine which has a methyl grou attached to the sulfur. *hus methionine is more hydro hobic, sterically larger and much less reacti+e than cysteine. Cysteine can be easily o,idi-ed to form a dimer containing disulfide bridge between two cysteines. )uch dimer is known as cystine (Fig. 2) and this feature is +ery im ortant for analysis the rimary structure of roteins, for effects on changes in secondary structure and for stabili-ation tertiary and .uaternary structure. )ulfhydryl grou of cysteine can be considered also as a +ery strong reducing factor, which is +ery im ortant for acti+ity of many roteins, a oligo e tides (glutathione), has a strong influence on redo, otential of en+ironment in certain com artments in +i+o, and can form a center of the acti+e site of some en-ymes (e.g. thiol roteinases). The presence of sulfhydryl group where hydrogen can be easily replaced by radicals and other groups, makes it possible to form a covalent bond with the other molecules. Cystine formation is an example of such activity. If a sulfur atom is bonded to sulfur atom of another cysteine, a covalent disulfide bridge is formed which is weaker (easier to split) than peptide bond, but stronger than any interaction of the other type (hydrogen bonds, salt bridges, hydrophobic and van der aals interactions). The easiness of formation of such covalent bond depends on overall redox potential of environment as well as p! (at low p! the e"uilibrium is shifted to the reduced, #$! form, under more basic conditions the #$! group is more willing to be oxidi%ed and replaced by #$&, where & is anything except for hydrogen).

CONTRIBUTION OF CYSTEINE IN PRIMARY STRUCTURE IN PROTEINS &ccording to the con+entional !inderstrom"!ang criteria (!inderstrom" !ang, 1/021 !inderstrom"!ang 2 )chellman, 1/0/) we can s ecify four general le+els of rotein structure3 " rimary structure " secondary structure " tertiary structure " .uaternary structure #n this hierarchy rimary structure is defined as the number and se.uence of amino acid residues linked together by e tide bonds, from %"terminus to C"terminus. &t resent there are listed si, general le+els of rotein structure (Cantor 2 )chimmel, 1/401 Creighton, 1//5) 3 " rimary structure " secondary structure " su ersecondary structure " domains " tertiary structure " .uaternary structure where rimary structure concerns amino acid se.uence including ost" translational modifications and disulfide bridges. *he meaning of rimary structure was then e,tended to to ology of disulfide bridges in roteins.

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*he to ology attern of disulfide bridges is a +ery useful tool for identification rotein families and su erfamilies with res ect to their common ancestry as well as their biological function. & e,am le of this is resented on Fig. 5 where roteinase inhibitors from different biological sources were di+ided into grou s of identical disulfide to ology. *his is es ecially useful in searching the related roteins of low le+el of o+erall homology in rimary structure. CONTRIBUTION OF CYSTEINE IN SECONDARY STRUCTURE IN PROTEINS )econdary structure of roteins is de endent on amino acid se.uence. #n that effect there are included side chains of amino acids and eriodicity of their occurrence along the oly e tide chain. For that reason we can admit some residues as heli, stabili-ers or heli, 6breakers6, etc. For heli, formation it is im ortant that e+ery 7th residue is of the same character according to it's hydro hobicity, and sterically acce table (no steric hindrance). )trongly hydro hobic amino acids, such as !eu, #le, 8al are considered to be heli, stabili-ers in many rediction algorithms. Cysteine which is not in+ol+ed in disulfide bridge formation can also stabili-e al ha heli,, although high reacti+ity of ")9 grou can ossibly distort the regular structure by interaction with the other reacti+e grou s. *he situation can dramatically change when cysteines in helical fragment form disulfide bridges. *his may im ly conformational changes which don't allow the fragment to acce t the helical regularity since disulfide bridges are stronger than the forces stabili-ing al ha heli,. &s an e,am le let's consider two small roteins re resenting o situations. osite

Rop rotein has a +ery regular structure, it consists of two al ha helices linked by turn. the only two cysteine residues are located far from each other and do not interact with each other. C=*#"# does not show any long helical fragment. *he entire secondary structure is rather irregular and +ery rigid because of disulfide cli s. #t is im ortant to note that the statistical algorithm of (arnier et al. (1/>4) redicts for C=*#"# significant contribution of helical fragments (Fig. 4 and /) which is absolutely not true. *he reason of such inconsistency is that the algorithm does not recogni-e the difference between cysteine and cystine and does not take the effect of )") bridges on secondary structure under consideration. ?robably, if all cysteine residues were in the reduced form (with free sulfhydryl grou s) high ercentage of helical fragments would be ossible. (f course similar effects of disulfide )clipping) on secondary structure can be expected in case of every type of secondary structure. Therefore, algorithms which do not recogni%e cysteine and cystine as separate units, cannot have high predictive accuracy, and may give results far from the actual situation. CONTRIBUTION OF CYSTEINE IN TERTIARY AND QUATERNARY STRUCTURE IN PROTEINS *he role of cysteine in tertiary structure of roteins is ob+ious. *he disulfide bridges formed by these residues link the fragments within a oly e tide chain, sometimes located +ery far from each other with res ect to their rimary structure. *hus, the cysteine residues lay the crucial role in the final three dimensional conformation of the rotein molecule, and make it stable (see Fig. >). @isulfide bridges are also in+ol+ed in the formation of the loo s of the backbone. *hese loo s can be e, osed on the surface of the molecule (far from the core) and a+ailable for the other factors to interact with them. *his is im ortant for the fragments containing residues in+ol+ed in recognition mechanism. @isul hide bonds im ro+e rigidity of the rotein. #t has +ery significant meaning in cysteine"rich roteins such as roteinase inhibitors, where e+en clea+age of the reacti+e site e tide bond doesn't change it's o+erall conformation and such 'modified' inhibitor still ossesses anti roteinase acti+ity (Fig. >).

:ne of them is transcri tion associated Rop rotein (re resor of rimer fro ;.coli) (Fig. <) which has two cysteine residues not in+ol+ed in )") formation. *he other one is C=*#"# (Fig. >), the try sin inhibitor from s.uash, containing si, cysteine residues which form three disulfide bridges.

*he loo formation stabili-ed by disulfide bridges can be obser+ed in many different roteins. &n e,am le of that are immunoglobulins, in which light and hea+y chains ha+e two and four such loo s res ecti+ely. *hey are recogni-ed as re etiti+e domains in this class of roteins, and therefore they are classified as mosaic roteins. *he .uaternary structure ractically e,ists thanks to resence of cysteine related disulfide bridges linking se arate oly e tide chains. #n most roteins which consist of more than one oly e tide chain, the chain are ke t together by interchain disulfide bridges. *he sim le e,am le is insulin, where & and A chain are linked by two bridges between Cys&> " CysA> and between Cys&20 " CysA1/. &nother e,am le is chymotry sin. *his en-yme is con+erted from its -ymogen by limited roteolysis in a few laces. Chymotry synogen consists of a single long oly e tide chain, the acti+e en-yme contains two or three chains, according to the ste of acti+ation. *hese chains of acti+e en-yme could not e,ist together without cysteine disulfide bridges linking them. (enerally we could say then, that without cysteine residues .uaternary structure wouldn't e,ist. CYSTEINE IN BIOLOGICALLY ACTIVE OLIGOPEPTIDES AND PROTEINS ;,ce t for the structural ro erties of roteins, cysteine residues may also lay im ortant role in catalytic rocesses of en-ymes, in storage and adBusting the reduction otentials, they can be a source of sulfur assimilable in easy way, etc. & good e,am le of the essential role of cysteine in en-yme catalytic acti+ity are thiol roteinases. *his is one of four maBor classes of roteinases. *o this class belong such roteinases as bromelain, a ain, ficin and some cathe sins. *he common feature of these en-ymes is that they all ha+e cysteine residue as the central amino acid of catalytic site (Fig. 10). Cysteine is directly in+ol+ed in the formation of acyl intermediate with the substrate. *he o+erall mechanism of catalysis reaction by thiol roteinases is the same as in serine roteinases. Cysteine at osition 20 in a ain has the same function as serine at osition 1/0 in try sin, chymotry sin or other serine roteinases. *he transition state in catalysis by a ain is shown on Fig. 10.

The different role of cysteine is in tripeptide # glutathione (+ig. ''). It is present in the e"uilibrium of two forms # reduced and oxidi%ed. The reduced form serves as )sulfhydryl buffer) that maintains the cysteine residues of hemoglobin and other erythrocyte proteins in the reduced state. It also works as detoxitant by reacting with hydrogen peroxide and organic peroxides. It protects red cells from oxidative damage. ,lutathione is involved in maintaining the proper e"uilibrium of the reduced form of the other reduction potential carrier # -./0!. Thus, the presence and state of glutathione is essential for the activity of many dehydrogenases (for example glutathione reductase). SO WHY CYSTEINE IS SPECIAL? 1. 2. 5. 7. 0. <. >. 4. Aecause it has a +ery reacti+e sulfhydryl grou at its side chain. *his uts cysteine in s ecial osition that cannot be re laced or substituted by any other amino acid. Aecause disulfide bridges formed by cysteine residues are ermanent com onent of rotein rimary structure. Aecause to ology of disulfide bridges is the basis in analysis of homology in rotein rimary structure. Aecause it can change secondary structure, by steric constraints. Aecause cysteine related disulfide bridges are ermanent element for stabili-ation of the tertiary structure. Aecause, in most cases, interchain )") bridges are absolute condition for .uaternary structure to e,ist. Aecause cysteine is at the center of catalytic site of thiol en-ymes. Aecause it lays im ortant role as the essential unit of the low" molecular"weight factors in the o+erall redo, otential in the biological systems.