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PubMed1997

Bound water in the proton translocation mechanism of the haem-copper oxidases.

We address the molecular mechanism by which the haem-copper oxidases translocate protons. Reduction of O2 to water takes place at a haem iron-copper (CuB) centre, and protons enter from one side of the membrane through a 'channel' structure in the enzyme. Statistical-mechanical calculations predict bound water molecules within this channel, and mutagenesis experiments show that breaking this water structure impedes proton translocation. Hydrogen-bonded water molecules connect the channel further via a conserved glutamic acid residue to a histidine ligand of CuB. The glutamic acid side chain may have to move during proton transfer because proton translocation is abolished if it is forced to interact with a nearby lysine or arginine. Perturbing the CuB ligand structure shifts an infrared mode that may be ascribed to the O-H stretch of bound water. This is sensitive to mutations of the glutamic acid, supporting its connectivity to the histidine. These results suggest key roles of bound water, the glutamic acid and the histidine copper ligand in the mechanism of proton translocation.

Amino Acid Sequence↗
PubMed1998

The pyridoxal-5'-phosphate-dependent catalytic antibody 15A9: its efficiency and stereospecificity in catalysing the exchange of the alpha-protons of glycine.

13C-NMR has been used to follow the exchange of the alpha-protons of [2-(13)C]glycine in the presence of pyridoxal-5'-phosphate and the catalytic antibody 15A9. In the presence of antibody 15A9 the 1st order exchange rates for the rapidly exchanged proton of [2-(13)C]glycine were only 25 and 150 times slower than those observed with tryptophan synthase (EC 4.2.1.20) and serine hydroxymethyltransferase (EC 2.1.2.1). The catalytic antibody increases the 1st order exchange rates of the alpha-protons of [2-(13)C]glycine by at least three orders of magnitude. We propose that this increase is largely due to an entropic mechanism which results from binding the glycine-pyridoxal-5'-phosphate Schiff base. The 1st and 2nd order exchange rates of the pro-2S proton have been determined but we were only able to determine the 2nd order exchange rate for the pro-2R proton of glycine. In the presence of 50 mM glycine the antibody preferentially catalyses the exchange of the pro-2S proton of glycine. The stereospecificity of the 2nd order exchange reaction was quantified and we discuss mechanisms which could account for the observed stereospecificity.

Antibodies, Catalytic↗
PubMed1998

Electrical current generation and proton pumping catalyzed by the ba3-type cytochrome c oxidase from Thermus thermophilus.

Several amino acid residues that have been shown to be essential for proton transfer in most cytochrome c oxidases are not conserved in the ba3-type cytochrome c oxidase from the thermophilic eubacterium Thermus thermophilus. So far, it has been unclear whether the Th. thermophilus ba3-type cytochrome c oxidase can nevertheless function as an electrogenic proton pump. In this study, we have combined charge translocation measurements on a lipid bilayer with two independent methods of proton pumping measurements to show that enzymatic turnover of the Th. thermophilus cytochrome c oxidase is indeed coupled to the generation of an electrocurrent and proton pumping across the membrane. In addition to a 'vectorial' consumption of 1.0 H+/e- for water formation, proton pumping with a stoichiometry of 0.4-0.5 H+/e- was observed. The implications of these findings for the mechanism of redox-coupled proton transfer in this unusual cytochrome c oxidase are discussed.

Amino Acid Sequence↗
PubMed1999

Effects of acute leptin administration on the differences in proton leak rate in liver mitochondria from ob/ob mice compared to lean controls.

In this investigation, the effects on proton leak of leptin administration to ob/ob mice was measured for liver mitochondria. We and others have shown that proton leak is approximately 3 times greater in liver mitochondria from ob/ob mice compared to lean controls at any given membrane potential. The results are consistent with obese mammals having higher lean mass-specific metabolic rates compared to lean controls. The increase in proton leak rate at any given membrane potential cannot be explained by the presence of free fatty acids associated with mitochondria isolated from the obese animals. The difference in proton leak must therefore represent a real difference in inner membrane permeability. Acute leptin (OB protein) administration restores the liver mitochondrial proton leak rate of ob/ob mice to that of lean controls. There was no effect on proton leak rate of liver mitochondria from sham-treated ob/ob mice. These novel results indicate a role for leptin, either directly or indirectly, in regulating the efficiency of oxidative phosphorylation.

Animals↗
PubMed2001

Sequential changes in MR water proton relaxation time detect the process of rat brain myelination during maturation.

For better understanding of the behavior of water molecules in the animal brain, changes in magnetic resonance water proton relaxation processes were studied in the rat during maturation. Midbrains of male Wistar rats were removed at various time points ranging from 2 to 70 days after birth. Changes in relaxation time (water proton longitudinal relaxation time by the inversion recovery, and water proton transverse relaxation time by the spin echo and the Carr-Purcell-Meiboom-Gill pulse sequence (CPMG)) and water content were then determined for various stages of brain development. During maturation both water proton longitudinal relaxation time and water proton transverse relaxation time values decreased and this finding paralleled the decline in water content. Using the CPMG pulse sequence, the transverse relaxation time values were observed to separate into two components after 21 days. Morphologically, the most prominent change at the matured stage of midbrain development in the rat is myelination. Water proton relaxation time, which can be estimated using the CPMG pulse sequence, showed a close correlation with myelination in the central nervous system.

Aging↗
PubMed2002

Proton transfer within the active-site cavity of carbonic anhydrase III.

The maximal turnover rate of CO2 hydration catalyzed by the carbonic anhydrases is limited by proton transfer steps from the zinc-bound water to solution, steps that regenerate the catalytically active zinc-bound hydroxide. Catalysis of CO2 hydration by wild-type human carbonic anhydrase III (HCA III) (k(cat) = 2 ms (-1)) is the least efficient among the carbonic anhydrases in its class, in part because it lacks an efficient proton shuttle residue. We have used site-directed mutagenesis to test positions within the active-site cavity of HCA III for their ability to carry out proton transfer by replacing various residues with histidine. Catalysis by wild-type HCA III and these six variants was determined from the initial velocity of hydration of CO2 measured by stopped-flow spectrophotometry and from the exchange of 18O between CO2 and H2O at chemical equilibrium by mass spectrometry. The results show that histidine at three positions (Lys64His, Arg67His and Phe131His) have the capacity to transfer protons during catalysis, enhancing maximal velocity of CO2 hydration and 18O exchange from 4- to 15-fold compared with wild-type HCA III. Histidine residues at the other three positions (Trp5His, Tyr7His, Phe20His) showed no firm evidence for proton transfer. These results are discussed in terms of the stereochemistry of the active-site cavity and possible proton transfer pathways.

Binding Sites↗
PubMed1999

Relative biological effectiveness of proton beams in clinical therapy.

PURPOSE: In clinical proton beam radiation therapy, an RBE of 1.1 relative to megavoltage X-rays is currently being employed at most treatment centers. This RBE pertains to radiation in the spread out Bragg-peak (SOBP) for all tissue systems, all dose levels per fraction and all proton beam energies. As the number of centers and treatment sites for which proton beam therapy continues to increase and additional experimental data is accrued, a re-assessment of the justification for a generic RBE is warranted. In this paper we address: (1) the constancy of the RBE along the central axis from the plateau entrance to the distal SOBP (upstream of the distal edge); (2) RBE as a function of dose (or cell survival level); and (3) the target cell or tissue (alpha/beta) dependency of the RBE. This analysis pertains to modulated proton beams of initial energies of approximately 70-200 MeV and SOBPs of approximately 2-10 cm, respectively. RESULTS AND CONCLUSIONS: With exceptions, the available experimental data indicate that the RBE of SOBP protons increases with decreasing dose or dose per fraction and increasing depth in the SOBP, with the magnitude of both effects likely being dependent on the alpha/beta ratios of the target cells or tissues. The use of a generic RBE of 1. for all tissues, especially those exhibiting low alpha/beta values such as CNS, may be too low, especially at dose levels of < or = 2 Gy/fraction. Systematic determination of the RBE values dependent upon the three interdependent variables identified in this manuscript (beam depth, dose size and target tissue) will provide an enhanced data base for detailed treatment planning and institutional trial comparisons, thereby maximizing the therapeutic benefit of proton beams.

Animals↗
PubMed2001

Effects of slow clinorotation on lipid contents and proton permeability of thylakoid membranes of pea chloroplasts.

Photochemical characteristics and lipid composition of thylakoid membranes from 12 day-old pea leaves that were exposed to slow clino-rotation were examined and compared with a vertical control. Proton permeability of thylakoid membranes was estimated from light-induced proton uptake (delta H+) and post-illumination proton efflux in chloroplast suspensions. The delta pH magnitude was calculated from the level of light-induced quenching of 9-aminoacridine fluorescence. Proton permeability of thylakoid membranes increased during exposure to clino-rotation. When subsequently transferred to darkness, proton efflux increased almost 2-fold in clinorotated leaves. The results were compared with data on pigment and polar lipid composition of photosynthetic membranes in clino-rotated and control plants. It was concluded that both the increase of proton permeability and the decrease of polar lipid content in chloroplasts were induced by clino-rotation.

Carotenoids↗
PubMed1998

The proton permeability of the inner membrane of liver mitochondria from ectothermic and endothermic vertebrates and from obese rats: correlations with standard metabolic rate and phospholipid fatty acid composition.

We measured the proton leak across the inner membrane of liver mitochondria isolated from six different vertebrate species and from obese and control Zucker rats. Proton leak at 37 degrees C was similar in rat and pigeon, and in obese and control Zucker rats. Compared to rat, it was lower in cane toad, shingleback lizard, and the Madeiran lizard Lacerta dugessi. Proton leak at 20 degrees C was similar in xenopus toad and higher in rainbow trout, compared to rat. In general, proton permeability and substrate oxidation activity were greater in liver mitochondria from endotherms than those from ectotherms. Analysis of this and previous data showed that proton leak per milligram of mitochondrial protein correlated with standard metabolic rate, and proton leak per milligram of inner membrane phospholipid correlated with 11 phospholipid fatty acid compositional parameters, including unsaturation index.

Animals↗
PubMed2000

Proton relative biological effectiveness (RBE) for survival in mice after thoracic irradiation with fractionated doses.

PURPOSE: This study aims at providing relative biological effectiveness (RBE) data under reference conditions accounting for the determination of the "clinical RBE" of protons. METHODS AND MATERIALS: RBE (ref. (60)Co gamma-rays) of the 200 MeV clinical proton beam produced at the National Accelerator Centre (South Africa) was determined for lung tolerance assessed by survival after selective irradiation of the thorax in mice. Irradiations were performed in 1, 3, or 10 fractions separated by 12 h. Proton irradiations were performed at the middle of a 7-cm spread out Bragg peak (SOBP). Control gamma irradiations were randomized with proton irradiations and performed simultaneously. A total of 1008 mice was used, of which 96 were assessed for histopathology. RESULTS: RBEs derived from LD50 ratios were found not to vary significantly with fractionation (corresponding dose range, approximately 2-20 Gy). They, however, tend to increase with time and reach (mean of the RBEs for 1, 3 and 10 fractions) 1.00, 1.08, 1.14, and 1.25 for LD50 at 180, 210, 240, and 270 days, respectively (confidence interval approximately 20%). alpha/beta ratios for protons and gamma are very similar and average 2.3 (0.6-4.8) for the different endpoints. Additional irradiations in 10 fractions at the end of the SOBP were found slightly more effective ( approximately 6%) than at the middle of the SOBP. A control experiment for intestinal crypt regeneration in mice was randomized with the lung experiment and yielded an RBE of 1.14 +/- 0.03, i.e., the same value as obtained previously, which vouches for the reliability of the experimental procedure. CONCLUSION: There is no need to raise the clinical RBE of protons in consideration of the late tolerance of healthy tissues in the extent that RBE for lung tolerance was found not to vary with fractionation nor to differ significantly from those of the majority of early- and late-responding tissues.

Animals↗
PubMed2002

Preclinical biological assessment of proton and carbon ion beams at Hyogo Ion Beam Medical Center.

PURPOSE: To assess the biologic effects of proton and carbon ion beams before clinical use. METHODS AND MATERIALS: Cultured cells from human salivary gland cancer (HSG cells) were irradiated at 5 points along a 190 MeV per nucleon proton and a 320 MeV per nucleon carbon ion beam, with Bragg peaks modulated to 6 cm widths. A linac 4 MV X-ray was used as a reference. Relative biologic effectiveness (RBE) values at each point were calculated from survival curves. Cells were also irradiated in a cell-stack phantom to identify that localized cell deaths were observed at predefined depth. Total body irradiation of C3H/He mice was performed, and the number of regenerating crypts per jejunal section was compared to calculate intestinal RBE values. For carbon ion and referential 4 MV X-ray beams, mouse right legs were irradiated by four-fractional treatment and followed up for skin reaction scoring. RESULTS: RBE values calculated from cell survival curves at the dose that would reduce cell survival to 10% (D10) ranged from 1.01 to 1.05 for protons and from 1.23 to 2.56 for carbon ions. The cell-stack phantom irradiation revealed localized cell deaths at predefined depth. The intestinal RBE values ranged from 1.01 to 1.08 for protons and from 1.15 to 1.88 for carbon ions. The skin RBE value was 2.16 at C320/6 cm spread-out Bragg peak (SOBP) center. CONCLUSION: The radiobiologic measurements of proton and carbon ion beams at Hyogo Ion Beam Medical Center are consistent with previous reports using proton beams in clinical settings and carbon ion beams with similar linear energy transfer (LET) values.

Animals↗
PubMed2003

Another approach to protons with constricted mobility in white matter: pilot studies using wideline and high-resolution NMR spectroscopy.

We report a new approach for the identification of an independent method of studying the semi-solid pool of protons, i.e., protons with constrained motion as a result of being bound to lipid and protein matrices. These protons cannot be observed using conventional imaging techniques since their transverse relaxation times are much shorter than the minimum echo times that are currently available on clinical scanners. In this pilot study, in vitro multicomponent transverse relaxation experiments were made on human white matter slices, fixed in formalin (7 normal and 5 with multiple sclerosis). The transverse relaxation decay curves were multiexponential and were decomposed to yield three primary components. The shortest T(2) component that we obtained (a component too short to be seen by in vivo methods) was of the order of microseconds. We hypothesize that this might correspond to the macromolecular pool of lipid protons trapped within the myelin sheaths. To our knowledge, this is the first attempt at extracting this ultra short T(2) component from human white matter. Subsequently, an attempt was made to directly detect the lipid protons in a proton NMR spectrum and, if possible, measure their concentration in some of the tissues, using the technique of magic angle spinning.

Brain↗
PubMed1999

Proton radiobiology and uncertainties.

This paper briefly reviews proton radiobiology. Clinical applications of protons produced by accelerators have led to a significant biological literature that contributes to our goal of estimating the proton shielding requirements for human interplanetary missions. Protons are primarily a low-LET radiation with biological effects much like gamma radiation. There are however data indicating enhanced biological effectiveness for small doses of very low energy (<10 MeV) stopping protons, and some limited data for extremely high energy protons (>0.5 GeV).

Animals↗
PubMed2002

Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase: theoretical and experimental study of the effect of glutamic acid 284 on the protonation state of lysine 213.

The crystal structure of Escherichia coli phosphoenolpyruvate (PEP) carboxykinase shows Lys213 is one of the ligands of enzyme-bound Mn2+ [Nat. Struct. Biol. 4 (1997) 990]. The direct coordination of Mn2+ by N(epsilon) of Lys213 is only consistent with a neutral (uncharged) Lys213, suggesting a low pKa for this residue. This work shows, through theoretical calculations and experimental analyses on homologous Saccharomyces cerevisiae PEP carboxykinase, how the microenvironment affects Mn2+ binding and the protonation state of Lys213. We show that Glu284, a residue close to Lys212, is required for correct protonation states of Lys212 and Lys213, and for Mn2+ binding. deltaG and deltaH values for the proton reorganization processes were calculated to analyze the energetic stability of the two different protonation states of Lys212 and Lys213 in wild-type and Glu284Gln S. cerevisiae PEP carboxykinase. Calculations were done using two modeling approaches, ab-initio density functional calculations and free energy perturbation (FEP) calculations. Both methods suggest that Lys212 must be protonated and Lys213 neutral in the wild-type enzyme. On the other hand, the calculations on the Glu284Gln mutant suggest a more stable neutral Lys212 and protonated Lys213. Experimental measurements showed 3 orders of magnitude lower activity and a threefold increase in Km for Mn2+ for Glu284Gln S. cerevisiae PEP carboxykinase when compared to wild type. The data here presented suggest that Glu284 is required for Mn2+ binding by S. cerevisiae PEP carboxykinase. We propose that Glu284 modulates the pKa value of Lys213 through electrostatic effects mediated by

Amino Acid Sequence↗
PubMed2000

Proton-coupled structural changes upon binding of carbon monoxide to cytochrome cd1: a combined flash photolysis and X-ray crystallography study.

We have investigated dynamic events after flash photolysis of CO from reduced cytochrome cd(1) nitrite reductase (NiR) from Paracoccus pantotrophus (formerly Thiosphaera pantotropha). Upon pulsed illumination of the cytochrome cd(1)-CO complex, at 460 nm, a rapid (<50 ns) absorbance change, attributed to dissociation of CO, was observed. This was followed by a biphasic rearrangement with rate constants of 1.7 x 10(4) and 2.5 x 10(3) s(-1) at pH 8.0. Both parts of the biphasic rearrangement phases displayed the same kinetic difference spectrum in the region of 400-660 nm. The slower of the two processes was accompanied by proton uptake from solution (0.5 proton per active site at pH 7.5-8.5). After photodissociation, the CO ligand recombined at a rate of 12 s(-1) (at 1 mM CO and pH 8.0), accompanied by proton release. The crystal structure of reduced cytochrome cd(1) in complex with CO was determined to a resolution of 1.57 A. The structure shows that CO binds to the iron of the d(1) heme in the active site. The ligation of the c heme is unchanged in the complex. A comparison of the structures of the reduced, unligated NiR and the NiR-CO complex indicates changes in the puckering of the d(1) heme as well as rearrangements in the hydrogen-bonding network and solvent organization in the substrate binding pocket at the d(1) heme. Since the CO ligand binds to heme d(1) and there are structural changes in the d(1) pocket upon CO binding, it is likely that the proton uptake or release observed after flash-induced CO dissociation is due to changes of the protonation state of groups in the active site. Such proton-coupled structural changes associated with ligand binding are likely to affect the redox potential of heme d(1) and may regulate the internal electron transfer from heme c to heme d(1).

Binding Sites↗
PubMed2000

Sequence-specific DNA binding by the glucocorticoid receptor DNA-binding domain is linked to a salt-dependent histidine protonation.

We used isothermal titration calorimetry in the temperature range 21-25 degrees C to investigate the effect of pH on the calorimetric enthalpy (delta H(cal)) for sequence specific DNA-binding of the glucocorticoid receptor DNA-binding domain (GR DBD). Titrations were carried out in solutions containing 100 mM NaCl, 1 mM dithiothreitol, 5% glycerol by volume, and 20 mM Tris, Hepes, Mops, or sodium phosphate buffers at pH 7.5. A strong dependence of delta H(cal) on the buffer ionization enthalpy is observed, demonstrating that the DNA binding of the GR DBD is linked to proton uptake at these conditions. The apparent increase in the pK(a) for an amino acid side chain upon DNA binding is supported by the results of complementary titrations, where delta H(cal) shows a characteristic dependence on the solution pH. delta H(cal) is also a function of the NaCl concentration, with opposite dependencies in Tris and Hepes buffers, respectively, such that a similar delta H(cal) value is approached at 300 mM NaCl. This behavior shows that the DNA-binding induced protonation is inhibited by increased concentrations of NaCl. A comparison with structural data suggests that the protonation involves a histidine (His451) in the GR DBD, because in the complex this residue is located close to a DNA phosphate at an orientation that is consistent with a charged-charged hydrogen bond in the protonated state. NMR spectra show that His451 is not protonated in the unbound protein at pH 7.5. The pH dependence in delta H(cal) can be quantitatively described by a shift of the pK(a) of His451 from approximately 6 in the unbound state to close to 8 when bound to DNA at low salt concentration conditions. A simple model involving a binding competition between a proton and a Na(+) counterion to the GR DBD-DNA complex reproduces the qualitative features of the salt dependence.

Animals↗
PubMed1993

Acid multimers of oligodeoxycytidine strands: stoichiometry, base-pair characterization, and proton exchange properties.

The structure recently proposed for the acid form of the oligonucleotide 5'-d(TC5) is a four-strand "tetrad" in which two parallel-stranded, base-paired duplexes are intimately associated, with their hemiprotonated C-C+ base pairs face-to-face and fully intercalated, in a so-called "i-motif" (Gehring et al., 1993). We use the amino and imino proton spectra to establish the structure and symmetry of the base pairs, properties which are a primary element in the resolution of the acid form describe above. The amino proton spectrum gives the best lower limit (8 x 10(4) s-1) on the rate of the imino proton jumping process which is responsible for the base-pair symmetry. The stoichiometry of the acid form of other deoxycytidine sequences is studied by gel filtration chromatography and in one case by an NMR equilibrium titration. In all cases, i.e., d(C12), d(T2C8T2), d(C4TC4), d(TC5), d(C5), d(C4), d(TC4), d(TC3T), and d(TC3), the acid form elutes as a tetramer. A single-strand component is also present in some cases. But no dimer is observed, except for some samples prepared by quenching from high temperatures. The characteristic H1'-H1' interresidue NOESY cross-peaks of the d(TC5) structure (Gehring et al., 1993) are also found in all the tetramers where they have been searched for, i.e., those of d(T2C8T2), d(C4TC4), d(TC3T), and d(TC3) (not shown), suggesting that these tetramers also are built on the i-motif and that such structures may be formed generally by strands containing a stretch of as little as three deoxycytidines. From the NMR titration of d(TC3), we derive a free energy of -7.6 kJ/mol per cytidine base pair for the formation of the tetramer from single strands. The free energy released by packing a base pair into the i-motif is comparable to that released in forming the base pair itself. Imino proton exchange is limited by base-pair opening, thanks to efficient intrinsic exchange catalysis: this explains the lack of effect of added catalysts. The base-pair lifetime is hundreds of times longer than in any DNA duplex, presumably due to the base-pair intercalation geometry. The variation of the lifetime along the sequence of the d(TC5) tetramer provides support for the recently proposed structure. The internal amino proton exchanges from the open state of the C-C+ pair, at a rate compatible with a pK of 9 appropriate for C+. But the external proton exchanges from the closed state, as with a pK of 17!(ABSTRACT TRUNCATED AT 400 WORDS)

Base Composition↗
PubMed1993

Effect of the arginine-82 to alanine mutation in bacteriorhodopsin on dark adaptation, proton release, and the photochemical cycle.

The pH dependence of the rate constant of dark adaptation (thermal isomerization from all-trans- to 13-cis-bR) drastically changes when Arg82 of bacteriorhodopsin is replaced by an alanine. In the wild type (WT) the rate decreases sharply between pH 2.5 and pH 5. In R82A the sharp decrease is shifted to pH > 7. This correlates with the shift in the pK of the purple-to-blue transition from pH 2.6 in the wild type to pH 7.2 in the mutant (in 150 mM KCl). We propose that the same group that controls the purple-to-blue transition, namely, Asp85, catalyzes dark adaptation. The rate of dark adaptation in the R82A mutant is proportional to the fraction of protonated Asp85, indicating that dark adaptation occurs when Asp85 is transiently protonated. Thermal isomerization is at least 2 x 10(3) times more likely when Asp85 is protonated (blue membrane) than when it is deprotonated (purple membrane). The pH dependence of dark adaptation in the WT can be explained by a model in which the rate of dark adaptation in the WT is also proportional to the fraction of protonated Asp85 and that the pK of Asp85 depends on some other group, X, which deprotonates (or moves away from Asp85) with pK9 and causes the shift in the pK of Asp85 from 2.6 to 7.2. The quantum yield of light adaptation is at least an order of magnitude less in R82A as compared to the WT. The rise time of M formation is very fast in R82A and, unlike the WT, pH independent (1 microsecond versus 85 and 6 microseconds in the WT at pH 7 and 10, respectively). The activation energy of the L to M transition is 6.9 kcal/mol versus 13.5 kcal/mol in the WT. Thus the loss of a positive charge in the active site greatly increases the rate of light-induced deprotonation of the Schiff base. In the R82A mutant, the M decay at pH > 8.8 is much faster than the recovery of initial bR, which suggests a decrease in the rate of back-reaction from N to M. In a suspension of R82A membranes the rate of proton release as measured by the pH-sensitive dye pyranine is delayed by at least 20-fold (in 2 M KCl), while the uptake of protons did not change much (12 ms in the WT versus 8 ms in R82A).(ABSTRACT TRUNCATED AT 400 WORDS)

Alanine↗