Hydrolysis of Phosphomonoesters Study

Introduction

Site-directed mutagenesis was the primary basis of this portion of the laboratory directed toward determining the active site of the enzyme catalysis for hydrolysis of phosphomonoesters. The criteria was to turn the original sequence with Arginine 166 into Glutamine 166 through a point mutation. The primer location is located in the 401511 sequence of the phoA gene of alkaline phosphatase in E. Coli.

The primer sequence is: 5′- G CTG GTG GCA CAT GTG ACC TCG CGC AAA TGC TAC GGT CCG AGC -3’.

The mutated primer sequence is: R166Q1 5’-G CTG GTG GCA CAT GTG ACC TCG CAG AAA TGC TAC GGT CCG AGC-3’.

The reverse complementary mutated primer sequence is: R166Q2 5’-GCT CGG ACT CGG ACT GTC GCA TTT CTG CGA GGT CAC ATG TGC CAC CAG C-3’.

A calculation for the melting temperature can be determined by the following equation:

TM= 81.5 + 0.41 (%GC) – 675/N- % mismatch

The TM value for Gln-166 was determined to be 85.95ËšC. It is necessary for the TM value to be above 75ËšC for successful mutation.

Running on the null hypothesis, it would be clear that nothing would change for the kinetic parameters. If something were to change, the kinetic parameters for the Michaelis constant (Km), which determine the velocity of an enzyme as well as calculated to be ½ the Vmax, would be hypothesized much higher after the mutation to glutamine since the mutation allows for 61% more accuracy from arginine’s CGG at 25% to glutamine’s CAG at 86%. The increase in the binding affinity creates the hypothesis that since it is bound closer together creating a stronger binding affinity, the maximal speed (Vmax) would be reduced since it does not require as much conversion between substrate and product.

Mechanism of Alkaline Phosphatase

Alkaline phosphatase (APase) is an enzyme located in the periplasmic space of E. Coli. The structure of APase, usually in the form of a dimer, shows two zinc metallonzymes and a magnesium ion in the active center. There is a bridging ligand with the protein Asp51 in the active center for the two zinc ions and the one magnesium ion. There are four phosphate oxygens; two phosphate oxygens form a phosphate bridge between the two zinc ions and the other two form hydrogen bonds with the guanidinium group of arg-166 (Coleman, 1992). The dimer is two identical subunits that each contain 429 amino acids. (Coleman, 1992). The most important amino acids located at the active site of APase are the four Cysteine residues represented as combined intrachain disulfides. The phosphorylated residue was Ser 102, which is phosphorylated during phosphate hydrolysis, to begin the nucleophilic attack on phosphorus. Each monomer contains a leucine rich environment of 22 residues. (Coleman, 1992). The first zinc ion is necessary in activating the leaving group of the ester oxygen in order for Ser 102 to phosphorylate (Coleman, 1992). The second zinc ion is necessary for the ester oxygen of the serylphosphate to begin activation of the phosphoseryl intermediate during hydrolysis of Ser 102. When the phosphoseryl intermediate is formed during the first zinc formation, a hydroxide is formed. (Coleman, 1992).

Figure 1: this figure represents the monomer of alkaline phosphatase showing the zinc triad with magnesium in the active center. (Coleman, 1992).

The proposed mutation as discussed, is mutating the arg-166 to glu-166. It is speculated that there will be no effect on the enzyme during the mutation. Arg-166 is located in the guanidinium group and is an electrophilic species while APase is being phosphorylated. Arg-166 is speculated to play a role in stabilizing the developing negative charge on the oxygen of the leaving group, pentacoordinate transition state, or help bind the phosphate group. As discovered byButler-Ransohoff et al, it is discovered that Arg-166 has no effect on the hydrolysis of phosphate monoesters and therefore will not be effected after mutation.

Competent Cells

Competent cells are used to help the DNA get into the cell. DNA is negatively charged and the calcium ions from the competent cells of DH5-α cells are positively charged. The purpose of calcium chloride’s positive ions is to create an equilibrium between negative charge on the cell membranes phospholipid heads and the phosphate group on the plasmid DNA. This is where the genetic modification occurs. Therefore, they help by binding to the DNA and move it into the cell after the cell goes through a heat shock cycle. Competent cells have a very high rate for transformation efficiency. The proposed understanding of how this works, is that the calcium ions are positive and therefore weaken the electrostatic repulsion, which in turn weakens the cell walls. When the cell goes through heat shock, it causes the pressure to increase opening the cell and allowing the competent cells to bring the DNA into the cell by allowing pores to be created with the weakened cell wall. This forces the plasmid to become supercoiled so that it can pass through the pores created by the charge difference and heat shock. The competence comes from the cold bath of DH5-α cells in calcium chloride which shocks the cells causing pores and then heat the cell to 42ËšC for roughly 2 minutes, more than this could cause the cell membrane to denature and the pEK-154 mutated plasmid would become denatured.

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DH5-α is a strain from E. Coli commonly used in laboratory practices due to it having the phoA- gene deficiency. This is necessary and desirable because it is easily transformed for creating competent cells. DH5-α cells are used because they are a common strain in E. Coli and are commonly used for cloning. In addition, they promote stability and help improve the quality of the plasmid when using the Miniprep kit. (Dagert, 1979).

A growth curve was created after incubation of the cells for competent cells. This was done to show the growth period during the lag phase is depicted by the highest point on the growth curve. This is taken to determine how much growth you have and is determined through a logarithm vs. time on a graph. The method used is optical density (OD) at 600nm to measure the transmittance on a spectrophotometer. If the value for the OD₆₀₀ is high, the protection factor by a filter is lower and vise versa. This method is used to determine how much light is absorbed through the bacterial cells.

Site-Directed Mutagenesis

The sequence chosen for the point mutation from Arginine 166 to Glutamine 166 was sequenced in Ann Arbor. Site-Directed Mutagenesis synthesizes two complimentary oligonucleotides double stranded DNA template with glutamine 166 mutation, which is tagged with unmodified nucleotide sequences to a single stranded DNA template to allow mutation to occur and then reforms the new double strand DNA template during thermal cycling. When this occurs, the newly single stranded DNA template forms complementary strands with the enzymes and nucleotides to produce a higher result of strands. The purpose of site-directed mutagenesis is to make specific changes to the DNA sequence of a gene. Quik Change Lightening Mutagenesis kit provides a faster and more reliable insertion of the mutation with a simple three step method.

The multiple cycles are subjected to a polymerase chain reaction (PCR) method. This allows multiple sets of mutated DNA to be produced within a short period of time. PCR works by heating and cooling the samples, since the double DNA is subjected to separation when heated it causes the srtands to separate allowing nicks in the template to occur.

pUC-18 is a control plasmid used for determining if a mutation was successful and efficient.

Figure 2: pUC-18 plasmid DNA genetically mutated to contain the LacZ gene as well as the ampicillin resistance gene. The polylinker cuts the section of the DNA by the Dpn1 restriction endonuclease creating a linear DNA strand allowing for the binding of the mutation of Arg-166 to Glu-166 to occur.

pUC-18 is a circular double stranded DNA molecule. The reason pUC-18 was chosen as the control positive plasmid is because it was genetically manipulated to contain an ampicillin resistance gene as well as a β-galactosidase enzyme known as LacZ. The lacZ gene is essential for DNA mutation because it contains a region to insert a polylinker, which recognizes the Dpn I restriction endonucleases during digestion causing the plasmid to become linear and bind to the mutated plasmid DNA that has also been cut with the Dpn I restriction endonuclease.

A pWhitescript is used as the positive control plasmid used for mutagenesis. The control plasmid is combined with pEK154 plasmid containing the phA gene of APase and the vector double stranded DNA. The two oligonucleotide primers are the opposite ends of the vector. pWhitescript has the stop codon TAA inserted that stops the β-galactosidase enzyme from producing. The two oligonucleotide control primers create a point mutation that turns the T residue of the stop codon to the C residue of the glutamine codon (Gln, CAG). That allows enzyme β-galactosidase to be produced after pWhitescript is subjected to mutagenesis.

There are two oligonucleotide with the mutation primer as discussed earlier. New DNA polymerase called Q1 enzyme was used for double stranded DNA template when combined with dNTP mix it extended the two-oligonucleotide primers as well as allowing the new plasmid DNA to construct.

PCR Cycling parameters were used during Quik Change Site-Directed Mutagenesis for the transformation of arginine to glutamine by the use of the Pfu Enzyme. As visible from table 1, the cycling parameters were set up for this specific reaction for a point mutation. After cycling, the non-mutated parental supercoiled double stranded (dsDNA) is digested.

Table 1: Quik Change Site-Directed Mutagenesis Parameters

The first step of the three step simple method for Quik Change Lightening Mutagenesis kit is Thermal cycling at 95ËšC for two minutes. During this time, the DNA template becomes denatured allowing the primers to be subjected to heat treatments, which denature the pEK-154 template DNA strand and synthesizes the primers to extend linearly and cause nicks with Pfu Fusion-based DNA polymerase, which are then sealed by components within the Pfu enzyme blend. This polymerase allows for exact replication of original template and does not disrupt the orientation. The original mutated pEK-154 was used as the supercoiled double stranded DNA template

Figure 1: during thermal cycling, the double-stranded DNA Template is nicked and subjected to point mutation for arginine 166 to glutamine 166 and then sealed with components in the Pfu enzyme blend.

In the second step of the kit the template digests the parental DNA strand with methylated and hemimethylated DNA using the enzyme Dpn I endonuclease and subjected to thermal cycling for the second segment. The un-mutated pEK-154 plasmid DNA is the parental DNA while the mutated pEK-154 is the plasmid DNA with the desired mutation. With the nicks, the mutated plasmid with the two oligonucleotide primers is mixed with Dpn I endonuclease for methylated DNA that targets the sequence 5’-Gm6ATC-3’. This sequence helps digest the pEK-154 parental DNA template and has not been introduced into the methylase enyme allowing methylation to not effect the DNA and therefore will not be digested with the parental DNA strand. Similarly, the un-mutated is also digested in Dpn I restriction endonuclease to be methylated.

Figure 2: Dpn 1 enzyme endonuclease digests the parental DNA template with methylated and hemimethylated allowing for single stranded DNA for transformation.

Step 3 is the last cycling step where transformation of the newly mutated glutamine 166 single-stranded DNA. The DNA is added to Dpn I restriction endonuclease, which forms it into linear DNA stranded DNA duplex forming a double stranded DNA for the newly synthesized mutated DNA. After the mutated vector DNA with the primers has been nicked it is transformed into CaCl₂ competent DH5-α cells. This transformation must occur to repair the nicks caused during cycles to separate the DNA strands. The pWs, which is used as the control DNA is also transformed with competent DH5-α cells to help repair the nicks.

Transformation

Control screening used X-gal and IPTG to determine if β-galactosidase activity. The newly mutated pEK-154 cells are screened for alkaline phosphatase activity. This helps screen for β-galatosidase enzyme in E. Coli is a Lac Z gene that codes for this enzyme. This enzyme is useful because it breaks the lactose into galactose and glucose.

A Lac operon contains an operator and a promoter, which binds RNA-polymerase that starts transcription for the Lac I gene for I protein which can only bind to operator or lactose, but not both. When I protein is bound to lactose, in this case Isopropyl-1-β-D-galactopyranoside (IPTG) which mimics the structure of lactoase allowing RNA-polymerase to bind to the promoter to produce β-galactosidase. 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal), which is a substrate that cleaves galactose in β-galactosidase to produce a blue color. If no β-galactosidase is present they turn white on the agar plates. This determines if the cells contain the mutated CAA codon for glutamine instead of the stop codon TAA of the unmutated protein.

5-bromo-4-chloro-3-indolylphosphate (BCIP) is used to determine if alkaline phosphatase is present. BCIP like X-gal is a substrate that cleaves the phosphate group off of alkaline phosphatase producing a blue color.