Term
| single sequential displacement reactions are reactions with a ternary complex. what does this mean? |
|
Definition
this means both substrates bind at once
these reactions can be
Random order reactions
or
Ordered reactions |
|
|
Term
| what is formed from a double displacement reaction? |
|
Definition
a substituted enzyme intermediate is formed
the mechanism is described as the ping-pong mechanism |
|
|
Term
| lactate dehydrogenase uses which substrate to convert pyruvate to lactate? |
|
Definition
| it uses NADH as a substrate |
|
|
Term
what are double displacement (ping pong) reactions characterised by?
what two products are formed in the double displacement reaction of aspartate and alpha-ketoglutarate?
which enzyme catalyses this? |
|
Definition
the formation of a substituted enzyme intermediate
the two products of aspartate and alpha-ketoglutarate are glutamate and oxaloacetate
this reaction is catalysed by aspartate aminotransferase
|
|
|
Term
| when can an uncompetitive inhibitor bind? |
|
Definition
| only after the substrate had bound |
|
|
Term
|
Definition
a proteolytic enzyme used for protein digestion in animals.
it cleaves peptide bonds selectively on C-terminal side of large hydrophobic amino acids (Try, Tyr, Phe, Met) |
|
|
Term
| how do irreversible inhibitors bind to the active site? |
|
Definition
|
|
Term
| how do group-specific reagents react? |
|
Definition
| they react with specific aa side chains eg -OH, -SH |
|
|
Term
| what are affinity labels? |
|
Definition
| these are substrate analogues that bind to the active site like S but then bind irreversibly and block the active site |
|
|
Term
| what are suicide inhibitors? |
|
Definition
| they are substrate analogues that are converted into an affinity label by the action of the enzyme eg 5-flurouracil |
|
|
Term
| in Chymotrypsin, which three amino acids form the Catalytic Triad? |
|
Definition
| Ser195, His57 and Asp102 form the catalytic triad |
|
|
Term
during catalysis, Ser195 becomes activated by which enzyme?
how do the catalytic triad function together? |
|
Definition
it becomes activated by His157
His157 polarises OH group of Ser195
It acts as a general base catalyst by accepting H+ ion
Ser becomes a highly reactive alkoxide ion (powerful nucleophile)
Asp102 assists by positioning His57 |
|
|
Term
| many proteolytic enzymes use a catlytic triad to acheive cleavge of the stable peptide bond using a similar mechanism. How many times has this triad evolved independantly? |
|
Definition
it has evolved independantly 3 times
Chymotrypsin and relatives eg, tyrpsin and elastase
Subtisilin-like proteases that are typified by bacterial subtisilin, but similar in other organisms too
Wheat Carboxypeptidase II and relatives - they use His and Cys rather than His and Ser |
|
|
Term
| what type of proteases are Chymotrypsin, trypsin and elastase? |
|
Definition
they are all serine-proteases
|
|
|
Term
| similarities and differences between three serine-proteases Chymotrypsin, trypsin and elastase |
|
Definition
they have around 40% amino acid sequence identity and almost identical 3D structures but Chymotrypsin cleaves after aa with large hydrophobic side (Try, Tyr, Phe & Met)
Trypsin cleaves after aa with long positively charged side chains (Arg & Lys)
Elastase cleaves after aa with small side chains (Ala & Ser) |
|
|
Term
| which amino acid is involved in the substrate specificity with the protease Trypsin? |
|
Definition
|
|
Term
| two aa are important in the substrate specificity of Elastase. what are they? |
|
Definition
|
|
Term
| which method can be used to dissect the importance of active site residues? |
|
Definition
Site-directed mutagenesis
the catalytic triad in subtilisin are each mutated to Alanine. Ser and His mutations < activity by 1 milion times btu Asp mutation < activity by 1/20,000 |
|
|
Term
subtilisin is used as an ....... in washing powders and other ...........
it can stand up well to presence of surfactants that might ........ many other enzymes.
problems included suseptibility to .... by bleaching agents. Inactivation at high ........ and ........ on substrate recognition
what has help over come these problems? |
|
Definition
enzyme, detergents, denature, oxidation, temperature, limitations
Mutagenesis has overcome each of these problems |
|
|
