Intro to Pharmacology and Toxicology Topics   

Kinases and Phosphatases

In mammals, protein kinases catalyze the formation of phosphomonoesters of Ser, Thr or Tyr using ATP as donor and usually Mg2+ as cofactor. Phosphatases catalyze the hydrolysis of phosphomonoesters. Some phosphates are stable, cannot be dephosphorylated by phosphatases.

Kinases and phosphatases are important because they seem to regulate many proteins. An estimated one third of all expressed proteins are phosphoproteins. Kinases themselves make up about 1-2% of the genome, while phosphatases make up 0.3-0.8%.

Some phosphorylations activate a protein, others deactivate it. Many proteins have multiple phosphorylation sites. The cell maintains a steady-state ration of phosphorylated to dephosphorylated protein.

Phosphorylation modifies protein structure and alters interactions with other molecules. This leads to changes in enzyme activity and important interactions with other proteins. Some protein kinases are themselves controlled by phosphorylation.

There are three major kinase gene families:

Catalytic subunits and other important residues are genetically conserved. The Ser/Thr/Tyr kinase family is comprised of cAMP-dependent kinases with highly conserved residues. They have a GXGXXG phosphate anchor region at the ATP binding site and an activation of T-loop, both in the active site. A mutation at the Lys79 position will inactivate the kinase. Sometimes a Ser or Thr change to Asp gives a constitutively active kinase. There are three classes in the Ser/Thr/Tyr kinase family: Ser/Thr kinase, Tyr kinase and dual specificity kinase (Ser/Thr/Tyr kinase)

There are three phosphatase families regarding their catalytic subunits: PPP, PPM and Tyr phosphatases. PPPs and PPMs are Ser/Thr phosphatase metalloenzymes, which act in a single step by activation of the metal ion with water or hydroxyl. They contain the conserved phosphodiesterase motif DXH(X)nGDXXDR(X)mDGNH(D/E). Some important PPPs are

Some important PPMs are:

Tyr phosphatases have a low molecular weight and may be tyrosine-specific (PTP) or have a dual specificity (Ser/Thr and Tyr). They form a cysteinyl-substrate intermediate and have a conserved motif: (I/V)HCXAGXXR(S/T)G.

The catalysis mechanism of tyrosine phosphatases follows two steps. First the phosphotyrosine protein undergoes nucleophilic attack by the cysteine sulfur, forming a covalent intermediary with the help of an aspartate for stability. Then a water molecule hydrolizes the intermediary with the help of Asp, Ser and Gln residues.

If any of the catalytic residues other than cysteine are mutated out, the enzyme-sustrate complex will not dissociate.

Continue to "Regulation and Substrate Recognition" or take a quiz: [Q1] [Q2] [Q3] [Q4].

Need more practice? Answer the review questions below (after sponsor).

1- What is the activity of kinases?

2- What is the activity of phosphatases?

3- What is the overall effect of phosphorylation on protein activity?

4- What is the effect of phosphorylation on a protein molecule?

5- Why are kinases and phosphatases important?

6- How much of the human genome is made of phosphoproteins?

7- List 3 major kinase gene families.

8- What kind og kinases belong to the Ser/Thr/Tyr kinases?

9- What are the major molecular features of Ser/Thr/Tyr kinases?

10- How can mutations change the activity of Ser/Thr/Tyr kinases?

11- List 3 clases of Ser/Thr/Tyr kinases

12- List 3 phosphatase families.

13- What is the activity and mechanism of action of PPP and PPM phosphatases?

14- List 7 PPP Phosphatases

15- What is another name for PP2B and how si it activated?

16- List 2 PPMs.

17- Where is pyruvate dehydrogenase phsphatase located?

18- How is PP2 activated?

19- List two types of tyrosine phosphatase

20- What kind of intermediary is formed by Tyr phosphatase?

21- Describe the two-step catalysis mechanism of Tyr phophatases.

22- What are the effects of mutations on Tyr phosphatase activity?

Continue scrolling to answers below (after sponsor).

Hey! DON'T PEEK!!! Finish the questions fist!

Answers:

1- What is the activity of kinases?
Catalyze the formation of phosphomonoesters of Ser, Thr or Tyr using ATP as a donor and usually Mg2+ as a cofactor.

2- What is the activity of phosphatases?
Catalyze the hydrolysis of phospjhomonoesters.

3- What is the overall effect of phosphorylation on protein activity?
Some phosphoryations activate proteins, others deactivate them. Many proteins have multiple phosphorylation sites. Some phosphates are stable and cannot be removed. The cell maintains a steady state ratio of phosphorylated to dephosphorylated protein.

4- What is the effect of phosphorylation on a protein molecule?
Modifies protein strucure and alters interactions with other molcules, leading to changes in enzymatic activity.

5- Why are kinases and phosphatases important?
They seem to regulate many proteins. One third of all expressed proteins are phosphoproteins.

6- How much of the human genome is made of phosphoproteins?
~ 1-2% kinases
~ 0.3-0.8 phosphatases

7- List 3major kinase gene families.
Ser/Thr/Tyr kinases
mitochondrial Ser/Thr kinases with bacterial His fold
DNA-protein kinases / TOR / FRAP / P14

8- What kind og kinases belong to the Ser/Thr/Tyr kinases?
PKAs with highly conserved residues

9- What are the major molecular features of Ser/Thr/Tyr kinases?
AGXGXXG phosphate anchor region at the ATP binding site, and an activation or T-loop, both at the active site.

10- How can mutations change the activity of Ser/Thr/Tyr kinases?
A mutation at the Lys72 position will inactivate the kinase. Sometimes a Ser or Thr changed to Asp gives a constitutively active kinase.

11- List 3 clases of Ser/Thr/Tyr kinases
Ser/Thr kinases
Tyr kinases
Dual-specificity kinases (Ser/Thr and Tyr)

12- List 3 phosphatase families.
PPP
PPM
Tyr phosphatases

13- What is the activity and mechanism of action of PPP and PPM phosphatases?
They are Ser/Thr metalloenzymes which act in a single step by activation of the metal ion with water or hydroxyl. They contain the conserved phosphodiesterase motif: DXH(X)nGDXXDR(X)mDGNH(D/E).

14- List 7 PPP Phosphatases
PP1
PP2A
PP2B
PP4
PP5
PP6
PP7

15- What is another name for PP2B and how si it activated?
Calcineurim is activated by Ca2+

16- List 2 PPMs.
PP2
pyruvate dehydrogenase phosphatase

17- Where is pyruvate dehydrogenase phsphatase located?
In the mitochondria.

18- How is PP2 activated?
By Mg2+

19- List two types of tyrosine phosphatase
Tyr-specific (PTP)
dual specificity (Ser/Thr and Tyr)

20- What kind of intermediary is formed by Tyr phosphatase?
A cysteinyl substrate covalent intermediary

21- Describe the two-step catalysis mechanism of Tyr phophatases.
Fisrt the phosphotyrosine protein undergoes nucleophilic attaack by the Cys sulfur, with the help of an Asp for stability. Then a water molecule hydrolyzes the intermediary with the hep of Asp, Ser and Gln residues.

22- What are the effects of mutations on Tyr phosphatase activity?
Ih any of the catalytic residues other than Cyd are mutatefd out, the enzyme-substrate complex will not dissociate.

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