Kinases and Phosphorylation

Kinases are like “presentation chefs” that embellish the protein products your “line cooks” (ribosomes) make - but they do charge - the “cherries” are bulky, negatively-charged phosphate groups, which can change not just what the protein looks like, but also how the protein acts. note: some kinases phosphorylate things other than proteins, but today I’m going to focus on the protein ones.

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Kinases modify proteins after they’re made (post-translationally) by transferring a phosphoryl (-PO₃²⁻) group to one of a protein’s charms, hence the term “phosphorylation.” Usually the phosphoryl group comes from the end of a nucleotide, most commonly from ATP. Although it’s technically a phosphoryl group being added (the 4th oxygen comes from the protein), we normally just say that kinases add phosphates. Phosphate (PO₄³⁻) has a central phosphorous(P) atom connected to 4 oxygen(O) atoms. It has “extra” electrons (e⁻) so it’s ➖ charged.  Putting so many negative charges right next to each other gives you an “unstable” high-energy molecule because the negative charges repel & it’s hard for the phosphate bonds to hold on. Enter the “escorts” - positively charged metals like Magnesium (Mg²⁺) complex with the phosphates & chaperone them as they move from place to place. And proteins like kinases that need to bind them have metals in their binding pocket as well as charged amino acids sticking into the binding pocket that can do a “changing of the guards.”  

Kinases are a type of enzyme - they catalyze (speed up) reactions without getting used up (they can add a phosphate to 1 protein then turn around & add 1 to another protein). Kinases catalyze the phosphoryl group transfer by helping hold together the reactants (ATP & protein substrate) in the right position for the transfer & stabilizing unstable intermediates. They can do this because the amino acid charms sticking out into their “active site” make a perfect environment for the hand-off. But how do they know where to add?  

Different kinases recognize different target consensus sequences - like their “dating profile” describing their “ideal match” sequence around the phosphorylation site - but there’s some wiggle room (& some are more promiscuous than others)(have higher specificity) - the ideality is due to that sequence making the substrate complement the shape, charge, etc. of their binding pocket (like maybe their binding pocket is negatively charged, so they want something with + charged amino acids near the phosphorylation site)(something you wouldn’t know from the linear sequence alone). But what’s the real “phosphorylation site?” which amino acid “charm” is actually getting changed? 

Only a few of the charms can be modified like this, the major ones being Tyrosine (Tyr or Y), Serine (Ser or S), Threonine (Thr or T) - what do they have in common? A hydroxyl (-OH) group that can ditch the hydrogen & act as a nucleophile & take that phosphoryl group to make a phosphate PO₄³⁻ group. Histidine (His or H) can also (less commonly) be phosphorylated, but on a Nitrogen (N)  

But under cellular conditions, there are plenty of H⁺ around (cellular pH is above their pKa), so these charms don’t really want to give up a H⁺ because that would make them negatively charged (-O⁻)… Enter the kinase! When the protein substrate binds, there’s a basic amino acid just waiting to pull it off. When it does, you get a strong nucleophile (the substrate’s -O⁻ really doesn’t like being negative & seeks out something that wants more electrons to share their extra with, so it attacks the γ phosphate conveniently sticking out right next to it). 

Hold on - didn’t you say phosphate has “extra” electrons and is - charged? Why does it want more? The phosphate group is negative overall BUT the central P is actually partly partly because the electronegative (electron-hogging) Os pull electrons (e⁻) away from it & metal or positively charged charms in the binding site can pull on this charge too which makes the P electrophilic(electron-loving) 

The exposed -O⁻ can therefore attack it. This forms a temporary pentacovalent intermediate (P bound to 5 Os) and then the bond between Β & γ phosphates is severed so you end up with ADP & a phosphorylated protein 

Now these products have fewer favorable contacts with the protein so they’re less tightly bound & can “fall out” so the kinase can do it all again but the enzyme has to “open up” to allow this (another conformational change) & this is often the slowest, “rate-limiting” step  

Many kinases can phosphorylate both Serine AND Threonine (which are both pretty small) but Tyr & His are bulky, so they require “more space” in the active site so they need different kinases. Thankfully, there are lots of different kinases & they do a lot of work. ~1/3 of all proteins in your cells likely have at least 1 phosphorylation & proteins can be phosphorylated at multiple sites.