Tyrosinase is a well-known copper-containing enzyme and is widely distributed in microorganisms, plants and animals. In fungi and vertebrates, tyrosinase catalyzes the rate-limiting step in the formation of the pigment melanin from tyrosine. In plants, the physiological substrates contain a variety of phenolics. Tyrosinase has been ascribed other functions apart from melanin production, including the detoxification of host plant defensive phenols for symbiotic bacteria [1,2], the sclerotisation of insect cuticles [3], and the synthesis of amino acid based antibiotics [4]. Recently, the activities of tyrosinase have been used in several biotechnological applications. Tyrosinase inhibitors are widely used in dermatological treatments and also applied in cosmetics. Actually, the specific tyrosinase inhibitors should be catalyzed by tyrosinase and form covalent bond with the enzyme, thus irreversibly inactivating the enzyme during catalytic reaction. Also, some chemical compounds reversibly bind to tyrosinase and reduce its catalytic capacity, and they could also be recognized as specific tyrosinase inhibitors. In general, some tyrosinase inhibitors exhibit only weak inhibitory effect due to their reactive and consumable properties toward tyrosinase or the quinone products. Several polyphenols, including flavonoids or stilbenoid, substrate analogues, free radical scavengers, and copper chelators, have been known to inhibit tyrosinase. Tyrosinase carries out the oxidation of phenols such as tyrosine and dopamine using dioxygen (O2). In the presence of catechol, benzoquinone is formed (see reaction below). Hydrogens removed from catechol combine with oxygen to form water.Tyrosinase (EC 1.14.18.1), a copper-containing enzyme, can cause enzymatic browning in raw fruits, vegetables,

and beverages.

It sounds as if what you are really looking for is not inhibitors in the normal sense but chemical modifiers - i.e. compounds that will reasonably specifically target a particular kind of amino acid and modify it covalently, literally taking out active enzyme molecules. If so, there are such reagents e.g. diethyl pyrocarbonate for His, tetranitromethane for Tyr, pyridoxal 5'-phosphate for Lys etc. However, each protein is unique and the fact that a reagent modifies an amino acid in free solution does not guarantee that it will also do so in the specific environment of your active site. So sometime you have to try out 3 or 4 different reagents to find the one that is most effective for your enzyme. Also, for the same reason, a reagent could end up unexpectedly modifying a different amino acid, so it is important to follow up with protein chemistry. You ask about concentrations and you need to experiment: what works in one case may me too dilute in another case. Each of these reagents has its own interesting properties and I would strongly recommend looking up a good book and following through to the original journal papers. An excellent guide is G.E. Means and R.E. Feeney, Chemical Modification of Proteins  and another one (this one in several volumes) is Lundblad and Noyes, Chemical Reagents for Protein Modification.

One final point: each of the amino acids you mention may occur 10 - 20 times in your protein.  A lysine reagent might modify not only your hypothetical active site lysine but also several others. You may be able to play with conditions, particularly pH, to make the modification more specific. Alternatively you may be able to use protection by substrate - i.e. you are only interested in the one (?) residue that is protected by substrate. The others will still be modified without decrease in activity.

He means to ask what are the probable inhibitors which bind these amino acids.

Dear Diptarka, by looking for inhibitors which beind to the amino acids

He means to ask what are the probable inhibitors which bind these amino acids.

Dear Diptarka, by looking for inhibitors which bind to the aminoacids you are not going to get any better idea.

Usually inhibitors bind to the clusters produced by these enzymes.  They may be the catalytic site or the other regions on enzyme structure.  To check if a molecule of interest binds you need to do what is called the insilico molecular docking. You have many softwares for this. To perform this you need 3D structure of your enzyme. Check if its available in protein database.  Check for any known inhibitors too and try the molecule which you are interested in. Then you can know where should the inhibitor or your compound binds so as to inhibit enzyme activity. Its not an easy job. Later to validate this result you gotta perform invitro and invivo studies.

Best

RR

@Raghu: I will carry out the in silico study myself. But, I need to validate it by wet lab. That is why I was asking for the same. Once, I have the structure of these inhibitors, I will carry out molecular docking and see if they bind to active site.

Designing inhibitors is big task. There are lot many virtual structure based docking softwares. There are some modes for designing inhibitors, that might help you before wet lab experiments also. First check if your enzyme is in protein database.

Best

RR

@Raghu: I have already modeled 3D structure of my enzyme in silico. Chemical modifiers are already in pubchem. So, I can carry out that. I am just a little bit unsure of the wet lab experiments to test.

Estimado amigo.

Los procesos de inhibición enzimática no son nada obvios. Como tampoco la ciencia misma. Mi humilde experiencia me ha indicado que la enzima AChE, enzima hidrolítíca bastante estudiada, tiene varios centros activos. Es una enzima compleja en vivo y vital en los seres vivos. Sin embargo, ella muestra un comportamiento similar en estado libre e immobilizada cuando es inhibida por carbamatos y organofosforados. Aun cuando los mecanismos de carbamilación y fosforilación son totalmente distintos. La cinética desinhibitoria, es también diferente. Más rápida en los carbamatos que en los organofosforados. En el caso de los carbamatos es competitiva y en los organofosforados acompetitiva. Yo demostré que no hay diferencias en el comportamiento de la AChE entre su forma libre y la inmobilizada.con los inhibidores mencionados, utilizando el acetato de para amino fenil como sustrato y siguiendo la actividad enzimática con un electrodo de carbón vítreo. los grupos amino de la Lisina fueron útiles para el proceso de inmobilización con glutaraldehido y BSA. Cada enzima es un mundo y hay que experimentar necesariamente con ellas para conocerlas.

Saludo cordial