ACJC 2023 H2 Bio Prelim P3 ANSWERs

This document consists of 21 printed pages. [Turn over Anglo-Chinese Junior College JC2 Biology Preliminary Examination Higher 2 CANDIDATE NAME FORM CLASS TUTORIAL CLASS INDEX NUMBER BIOLOGY Paper 3 Long Structured and Free-response Questions Candidates answer on the Question Paper. Additional Materials: Writing paper(s) 9744/03 29 August 2023 2 hours READ THESE INSTRUCTIONS FIRST Write your Name, Class and Index number in the spaces at the top of this page. Write in dark blue or black pen. You may use an HB pencil for any diagrams or graphs. Do not use staples, paper clips, glue or correction fluid. Section A Answer all questions. Section B Answer any one question on the separate writing paper(s) provided. The use of an approved scientific calculator is expected, where appropriate. You may lose marks if you do not show your working or if you do not use appropriate units. The number of marks is given in brackets [ ] at the end of each question or part question. At the end of the examination, fasten all the writing paper(s) used securely together. For Examiners’ use only Section A 1 / 30 2 / 10 3 / 10 Section B 4 or 5 / 25 Total / 75

2 Anglo-Chinese Junior College 2023 J2 H2 9744 Paper 3 Preliminary Examination For Examiner’s Use Section A Answer all the questions in this section. 1 Polyethylene terephthalate (PET) is one of the common polymers used to make plastic bottles. A bacterial species, Ideonella sakaiensis, was found to have the ability to break down PET through a two-step process catalysed by two enzymes. Fig. 1.1 shows the first step in which the enzyme PETase resulted in the degradation of PET into monohydroxylethyl terephthalate (MHET), and Fig. 1.2 shows the second step where the enzyme MHETase breaks down MHET into ethylene glycol (EG) and terephthalic acid (TPA). Fig. 1.1 Fig. 1.2 The structures of PETase and MHETase are shown in Fig. 1.3 and Fig. 1.4 respectively. Fig. 1.3 Fig. 1.4

3 [Turn over Anglo-Chinese Junior College 2023 J2 H2 9744 Paper 3 Preliminary Examination For Examiner’s Use (a) (i) With reference to Fig. 1.1 and Fig. 1.2, state the type of reaction that PETase and MHETase catalyse. 1. hydrolysis; …………………………………………………………………………………………. [1] (ii) Describe the secondary structures present in PETase and MHETase in Fig. 1.3 and Fig. 1.4. 1. Alpha helices and beta-pleated sheets can be found within the polypeptide; ………………………………………….…………………………………………………. 2. In an alpha helix, each N-H forms hydrogen bond with C=O four amino acids apart in the same polypeptide backbone; ………………………………………….…………………………………………………. 3. In beta -pleated sheets, each N-H forms hydrogen bond with C=O on adjacent part of the polypeptide backbone; ………………………………………….…………………………………………………. See hydrogen bond and backbone at least once. …………………………………………………………………...…………………….. [3]

4 Anglo-Chinese Junior College 2023 J2 H2 9744 Paper 3 Preliminary Examination For Examiner’s Use During the investigation of the rate of MHETase activity under different conditions, scientists attempted to find the most effective method to quench the enzyme activity (stop further enzymatic action) at the end of an experiment to accurately determine the true enzymatic rate. However, the solutions used for quenching the enzyme MHETase may also directly react with the substrate MHET and break the bonds in MHET. This unintended chemical reacti on gives rise to inaccurate determination of the enzymatic rate. Table 1.1 shows the results from using four different quenching methods to stop further enzymatic activity of MHETase. Table 1.1 quenching method proportion of MHET degraded due to quenching method and not the enzyme / % success in quenching enzyme activity Method 1: highly concentrated hydrochloric acid (6 M HCl) at 85 °C 39.40 cannot be determined, as high levels of products from the breakdown of MHET are found Method 2: 100% methanol at 85 °C 0.25 yes Method 3: 100 nM PMSF at 85 °C 1.30 no Method 4: pH 2.5 buffer solution at room temperature 0.00 no Methanol, used in Method 2, is an amphipathic solvent with the molecular formula, CH3OH. Fig. 1. 5 shows the structure of phenylmethylsulfonyl fluoride (PMSF), used in Method 3, which acts as an inhibitor that binds at the active site. Fig. 1.5

5 [Turn over Anglo-Chinese Junior College 2023 J2 H2 9744 Paper 3 Preliminary Examination For Examiner’s Use (b) (i) With reference to Table 1.1, identify the quenching method that should be used for the experiment and justify your answer. 1. *Method 2 / 100% methanol at 85 °C; ………………………………………….………………………………………………… 2. The proportion of MHET degraded due to the quenching method is low at 0.25%, which will allow for a more accurate determination of enzymatic rate; ………………………………………….…………………………………………………... 3. Method 2 also allowed for the enzyme activity to be quenched unlike the other three methods; ……………………………………………………………………………………………… ………………………………………………………………………………………….. [3] (ii) Explain the effect of dissolving an enzyme MHETase in an amphipathic solvent such as methanol. 1. The hydrophobic R groups of the amino acid residues in MHETase will interact with the hydrophobic CH 3 group on methanol via hydrophobic interactions; A! hydrophilic R groups of the amino acid residues in MHETase forms hydrogen bonds with the OH group on methanol instead of within itself ………………………………………….………………………………………………….. 2. This disrupts the bonds holding the 3D conformation of the enzyme together, causing denaturation; ……………………………………………………………………………………………… ……………………………………………………………………………………………… ………………………………………………………………………………………….. [2] (iii) Suggest why PMSF does not effectively quench the enzyme activity. 1. The substrate MHET concentration could be high enough to outcompete the competitive inhibitor; A! opposite phrasing – PMSF concentration is too low/lower than the substrate MHET concentration, hence PMSF is outcompeted ………………………………………….…………………………………………………... 2. The PMSF does not form permanent bonds with the active site; 3. Higher frequency of effective collisions between MHET and MHETase so / Enzyme-Substrate complexes can still form; ………………………………………………………………………………………….. [2]

6 Anglo-Chinese Junior College 2023 J2 H2 9744 Paper 3 Preliminary Examination For Examiner’s Use The scientists examined how proximity of the two enzymes , PETase and MHETase, influences overall rate of MHET degradation. Chimaeric proteins refer to two or more different proteins that are joined together artificially. Chimaeric proteins of MHETase and PETase were synthesised by using amino acid residues to covalently link the C terminus of MHETase to the N terminus of PETase, giving rise to MP8, MP12 or MP20 (see Table 1.2 and Fig. 1.6). Different linker lengths of amino acid residues resulted in varying mobility between the two enzymes. Table 1.2 chimaeric protein number of amino acid (aa) residues found in the linker MP8 8 MP12 12 MP20 20 Fig. 1.7 shows the rate of MHET degradation with PETase only, MHETase only, MP8, MP12 or MP20 added to the substrate MHET. Asterisks indicate statistically significant comparisons between MHETase only and each chimaeric protein with *P ≤ 0.01, **P ≤ 0.001, and ***P ≤ 0.0005. Fig.