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I am trying to attach a cross-linker to an enzyme and I am looking for candidates for reaction sites. I have several amino acids that are present on the surface of the enzyme (hydrophilic) but I am hoping someone can give some tips for amino acids that are easier to attach linkers to.

I originally was planning on attaching a linker to lysine because it is among the more reactive residues but my enzyme lacks a sufficient number of lysine groups.

Could any one give me some general advice on which amino acids would be better to investigate first? I know the specific residue I choose will depend mostly on the structure of the enzyme, but if you could give me a suggestion of which amino acids are typically easier to manipulate (ie. Lysine > glycine) I would greatly appreciate it.

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I'm assuming the OP concerns chemical means of modifying residues. Multiple protein residues other than Lysine may be labeled with an adduct. Here are some examples:

  1. Histidine residues may be labeled using an epoxide attached to a ligand. (Chen et al., 2003)

  2. Cysteine residues may be labeled with a nitrile derivative where the sulhydryl acts as the nucleophile. (Obella et al., 2007)

  3. Arginine residues may labeled using a derivative of the naturally occurring methylglyoxal. (Oya et al., 1999)

  4. Carboxylate functionality such as in Aspartate and Glutamate residues as well as the C-terminus may be labeled via the formation of an amide linker using a carbodiimide such as N-ethyl-3-N',N'-dimethylaminopropylcarbodiimide (EDC). (Gilles et al., 1999)

The above list of residues is not comprehensive, nor are the labeling agents. I recommend checking out the reviews by Basle et al. (2010) and Chen and Wu (2016).

References
Basle, E, Joubert, N, Pucheault, M (2010) "Protein Chemical Modification on Endogenous Amino Acids" Chemistry & Biology 17:213-227. DOI: 10.1016/j.chembiol.2010.02.008
Chen, X and Wu, Y-W (2016) "Selective chemical labeling of proteins" Org. Biomol. Chem. 14:5417-5439. DOI: 10.1039/C6OB00126B
Gilles, MA, Hudson, AQ, Borders, CL (1990) "Stability of water-soluble carbodiimides in aqueous solution" Anal. Biochem. 184:244-248. DOI: 10.1016/0003-2697(90)90675-Y
Gong Chen, Alexander Heim, Doris Riether, Dominic Yee, Yelena Milgrom, Mary Ann Gawinowicz, and Dalibor Sames (2003) "Reactivity of Functional Groups on the Protein Surface: Development of Epoxide Probes for Protein Labeling" J. Am. Chem. Soc. 125:8130–8133. DOI: 10.1021/ja034287m
Oballa, RM, Truchon, JF, Bayly, CI, Chauret, N, Day, S, Crane, S, et al. (2007) "A generally applicable method for assessing the electrophilicity and reactivity of diverse nitrile-containing compounds" Bioorg. Med. Chem. Lett. 17:998–1002. DOI: 10.1016/j.bmcl.2006.11.044
Oya, T, Hattori, N, Mizuno, Y, Miyata, S, Maeda, S, Osawa, T, Uchida, K (1999) "Methylglyoxal modification of protein. Chemical and immunochemical characterization of methylglyoxal-arginine adducts" J. Biol. Chem. 274:18492-18502. DOI: 10.1074/jbc.274.26.18492

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    $\begingroup$ If you try to modify one of the 20 naturally occurring amino acid, you have to make sure there is at least one on the surface, and that you don't have too many. In some instances, active site cysteines, serines, or histidines are more reactive than the same residues elsewhere on the surface (e.g. when they have a different $\mathrm{p}K_\mathrm{a}$ because of their surrounding). $\endgroup$
    – Karsten ♦
    Commented Feb 1, 2021 at 17:59
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    $\begingroup$ @KarstenTheis Indeed these are great points. I think the OP noted their concern about the lack of sufficient number of lysine groups. I inferred from this that their intent wasn't about targeting one specific residue, but a residue type that is sufficiently abundant for their experiments. I suspect the suggestion of using the EDC reagent may be reasonable in beginning to answer their question. $\endgroup$
    – z1273
    Commented Feb 1, 2021 at 18:11

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