Quiz 2 Enzyme

How to calculate the starting [MeCob]

A = elc

How to calculate initial rate

y = V0 = Vmax[S]/Km+ [S] OR V0 = slope/[Initial MeCob]

How to calculate kcat

Vmax/[Enzyme]

M1

Vmax

M2

Km

Calculate [Enzyme]

C1V1 = C2V2

[Starting MetH Stock]*[Volume Stock] = [MetH final in Assay]*(Vol Assay)

Explain the Quickchange method for site-directed mutagenesis

1. Design complementary forward+reverse primers coding for AA mut

2. PCR amplification - Primers + WT plasmid in thermocycling rxn, DNApol copies plasmid w Mut

3. RE DPN1

4. Transformation into Ecoli cells

5. Selection w ampicillin

6. Incubated - colonies grow

7. Protein synthesis. pCWG02 contains lac operon ahead of MetH (2-649) gene - IPTG induces protein exp - ZnCl2 added to ensure enough Zn binds to active site

Melting temp of Quickchange primers

> 78degC

Mutation must be placed where in a quickchange primer

middle

GC content of Quickchange primers

40% for stable annealing

How are primers designed for quickchange mutagenesis

First, you should obtain a plasmid DNA which contains the gene of interest. Primers would then be designed so that they are complementary to each other and contain a mutation. The primer will be complementary to the gene of interest, except the part where there is a mutation. This results in a hybrid of the original codon on the template strand and the mutated codon on the coding strand (so h bond won't form between them)

how are primers designed for sanger seq

Primer should be short (15-20 bp) and should NOT contain any

mutations

• Primer should anneal to only one place on the plasmid

• Primer should be 200 - 400 bp upstream of the region you mutated in

the plasmid, so the resulting sequence will be in the highest quality

area of the results

What does catalytic zn do (JC4)

Coordinates the sulfur of Hcy

What did JC4 researchers do

determined the crystal structures of substrate-free and Hcy-bound T. maritima MetE AND the crystal structures of a zinc-replete T. maritima N-terminal MetH fragment, in the absence and presence of Hcy,

What did researchers in JC4 observe in MetE

Inversion of Zn geometry enabled by conformational changes

MetE vs MetH

MetE: Cobalamin-independent MetH = Cobalamin-dependent

What did JC4 researchers find MetE and MetH have in common

Same active site core net charge

Surprising fact about Asn-234

Zn ligand in metH

JC4 potential roles of Zn inversion

1. Facilitating product release after methylation

2. May increase the nucleophilicity of Zn-thiolate -> methyl transfer

JC4 2 models for Zn inversion

1. Induced fit: binding of Hcy -> conformational changes that facilitate Zn displacement

2. Dynamic Zn: Zn oscillates btwn 2 tetrahedral geometries + substrate-induced conformational changes -> more elastic active site

What did researchers from JC5 do?

Determine crystal structure of the Fol module of T. thermophilus MS

key finding of JC5

T. maritima MS Fol module displays atypical and canonical beta8alpha 8 barrel, but the Fol module of T. thermophilus MS is a beta8 alpha7 barrel. Thus, the T. thermophilus MS Fol module represents the first described example of a noncanonical subclass of TIM barrels

descripe MTHF activation as discussed in JC5

MetH's Fol domain binds CH₃-H₄folate through a conserved hydrogen-bonding network that positions substrate for methyl transfer. Conserved AA residues, Asn and Asp residues, may create a chemical environment ideal for methyl transfer. The Fol domain contains a binding pocket that recognizes the pterin ring of MTHF. Comparing the Fol domain structures with and without CH₃-H₄folate revealed very little structural change - suggests a largely lock-and-key mode of substrate binding

MetH reactivation cycle

Cob(ii) is enzymatically reduced + methylated by AdoMet to regenerate MeCob cofactor

what did researchers in JC6 do

captured the full-length, high-resolution structure of MS in its apo-form and captured Cbl cofactor loading in crystallo.

what did researchers do to tMS in JC6

Purify its structure in the absence of cobalamin

tMS is...

highly heat tolerant (JC6)

Rather than relying onprotein:protein domain interactions, cobalamin....

appears to be the primary mediator governing which substrate-binding domain canform a ternary complex with the Cob domain, with linker regions andHis761 playing a role in guiding the conformation ensemble.

tMS shape (JC6)

compact wreath-like

Molecular juggling (JC6)

enzyme adopts different conformations to carry out homocysteine methylation, folate methylation, and cofactor reactivation.

__ change position when cobalamin binds.

His761 and Tyr1132

Role of His761 (JC6)

In the apo structure, this histidine faces outward, helping keep the cobalamin-binding pocket accessible. In the captured cobalamin-bound structure, it is not coordinated to cobalt; instead, it interacts with the phosphate tail of cobalamin's nucleotide component.

Role of Tyr 1132 (JC6)

This tyrosine moves over the upper face of the bound cofactor, acting like a lid.