Enzyme Kinetics 2

Which are true about bisubstrate reactions?

A transferase reaction is one where the functional group on one substrate is transferred to another substrate

In redox reactions, oxidising equivalents are transferred between substrates

In bisubstrate reactions with a sequential mechanism, all substrates must combine with the enzyme before the reaction can occur and products are released. Which of the following are also true of sequential bisubstrate reactions?

In Ping-Pong reactions, one or more products are released before all substrates have combined with the enzyme. Which of the following are also true of Ping-Pong reactions?

Pancreatic triacylglycerol lipase catalyses the hydrolysis of the esters of fatty acids into free esters and is involved in protein digestion.

Pancreatic triacylglycerol lipase is secreted by the pancreas and also found in saliva and the stomach

Free esters released from lipid digestion of fatty acids, are more soluble than fatty acids.

Pancreatic triacylglycerol lipase catalyses the hydrolysis of triacylglycerols at position 1 and 2 on the glycerol molecule, and formation of 1,2-diacylglycerols and 2-acylglycerols

Which are true about lipase?

What residues compose the catalytic triad of pancreatic triacylglycerol lipase?

The mechanism of triacylglycerol lipase: Asp [blank_start]H-bonds to His, to increase its basicity[blank_end]. His can then [blank_start]abstract a proton from Ser[blank_end] to increase its nucleophilicity. Then Ser is able to launch a nucleophilic attack on the [blank_start]ester carbonyl of the fatty acids[blank_end] (the glycerol backbone). This forms a [blank_start]tetrahedral intermediate[blank_end], which is stabilised by [blank_start]an oxyanion hole[blank_end] (with residues [blank_start]Phe and Leu[blank_end]). The negative charge collapses as [blank_start]His protonates the glycerol backbone[blank_end] and the carbonyl reforms, releasing the first product, [blank_start]1,2-diacylglycerol[blank_end]. His then abstracts a proton from a molecule of water, forming a reactive [blank_start]hydroxyl anion[blank_end]. [blank_start]This OH[blank_end] can than attack the [blank_start]carbonyl carbon of the lipid[blank_end], forming another intermediate, which is stabilised in an oxyanion hole. The negative charge collapses when [blank_start]His protonates Ser[blank_end], reforming the catalytic Ser and allowing release of a 2nd product, [blank_start]a free fatty acid[blank_end].

PLCA2 (phospholipase A2) requires a conformational change to function.

In PLCA2, the hydrophobic binding pocket is blocked by an inhibitor

The active site of PLCA2, consists of which of the following catalytic residues?

The active site contains a bound Mg.

PLCA2 mechanism: His is [blank_start]too far[blank_end] to activate for nucleophilic attack, so there is a [blank_start]second water[blank_end] which will later attack the scissile carbonyl carbon. Asp makes His more basic, His abstracts a proton from water. The activated water can nucleophilically attack the [blank_start]carbonyl carbon[blank_end], forming a tetrahedral intermediate. The role of Ca2+ is to coordinate the activated water molecule and eletrostatically stabilise negative charge on the oxygen of the tetrahedral intermediate ([blank_start]metal catalysis[blank_end]). Then there is the collapse of the tetrahedral intermediate. The [blank_start]His H-bonds[blank_end] to the hydroxyl group, causing it to abstract a proton from the lipid and reform water

Which are true of monotopic enzymes?

Which are true of multi-span enzymes?

The gram negative cell wall is less robust and lacks mechanical strength as there is less peptidoglycan.

LpxC is an essential zinc-dependent deacetylase of bacterial lipid A synthesis producing UDP-3-O-N-glucosamine. Which of the following are true about it?

1 proposed mechanism for LpxC action: [blank_start]Zn[blank_end] binds the active site. Glu acts as a base and [blank_start]deprotonates water[blank_end], making a good nucleophile to attack the [blank_start]carbonyl carbon[blank_end]. His stabilises the [blank_start]negative charge[blank_end] that develops. The negative charge collapses and [blank_start]Glu acts as acid[blank_end] and [blank_start]donates a proton back to[blank_end] the lipid. [blank_start]Acetic acid[blank_end] is also formed which loses a [blank_start]proton[blank_end] to form [blank_start]acetate[blank_end]. The product leaves and the Zn-lipid complex is displaced by water molecule and a new substrate.

Why is mechanism 1 for LpxC thought to be incorrect? What is the correct mechanism?

In terms of bacterial cell wall biosynthesis, which are true of transglycosylation?

To form peptidoglycans, enzymes exist to make trans additions of sugars. Which are true?

Transglycosylation mechanism of glycosyltransferases (GTs): Lipid binds to the membrane protein in a pocket and another lipid is fed through the membrane. The E114 residue acts as a [blank_start]bronsted base[blank_end], abstracting a proton from the [blank_start]4-OH[blank_end] group of the [blank_start]lipid II acceptor[blank_end]. Oxygen activates to act as a good nucleophile and can make attack on [blank_start]C1 position[blank_end] of sugar moiety. Glu E171 stabilises the negative charge forming (phosphate groups), coordinating [blank_start]pyrophosphate groups[blank_end] that form on the donor. Stabilisation is either directly or indirectly, mediated by a [blank_start]divalent metal cation (e.g. Mg)[blank_end] to stabilise negative charge. Upon attack on sugar, [blank_start]lipid-pyrophosphate[blank_end] leaving group can diffuse out of active site to give higher oligomeric state of lipid (through addition of more sugar to the incoming lipid). Must go from [blank_start]boat to chair[blank_end] conformation (because SN2 attack)

Mechanism for lipid II polymerisation by TG (transglycosylase): Glu acts as bronsted base and [blank_start]deprotonates 4-OH[blank_end] on [blank_start]GlcNAc[blank_end] of a lipid II molecule at the [blank_start]S1 site[blank_end]. This is followed by a simultaneous reaction with the C1 of another lipid II at S2 site with Glu. [blank_start]Lys and Arg[blank_end] stabilise the negative charge of phosphate and facilitate its diffusion as a leaving group. Then there is the [blank_start]transfer of the sugar and phosphate[blank_end] to growing chain. Essentially, the lipid II’s glycosyl [blank_start]donor site (S2)[blank_end] reacts with the acceptor site (S1) of lipid II to form a [blank_start]β1–4-[blank_end] linked glycan chain. Lipid keeps growing until certain length before leaving.