Amide Region Research Articles

Calculation of the infrared spectra of proteins.

The CHARMM22 force field with associated partial charges is used to calculate the infrared spectra of a number of small proteins and some larger biothreat proteins. The calculated high-frequency region, from about 2,500 to 3,500 cm(-1), is dominated by stretching modes of hydrogen bonded to other atoms, and is very similar in all proteins. There is a peak at 3,430 cm(-1) whose intensity is predicted by these calculations to be a direct measure of arginine content. The calculated low-frequency THz region, up to 300 cm(-1), is also very similar in all the proteins and just reflects the vibrational density of states in agreement with experimental results. Calculations show that the intermediate-frequency region between 500 and 1,200 cm(-1) shows the greatest difference between individual proteins and is also the least affected by water absorption. However, to match experimental measurements in the amide region, it was necessary to reduce the hydrogen partial charges.

Capsaicin inhibits collagen fibril formation and increases the stability of collagen fibers.

Capsaicin is a versatile plant product which has been ascribed several health benefits and anti-inflammatory and analgesic properties. We have investigated the effect of capsaicin on the molecular stability, self-assembly, and fibril stability of type-I collagen. It was found that capsaicin suppresses collagen fibril formation, increases the stability of collagen fibers in tendons, and has no effect on the molecular stability of collagen. Turbidity assay data show that capsaicin does not promote disassembly of collagen fibrils. However, capsaicin moderately protects collagen fibrils from enzymatic degradation. Computational studies revealed the functions of the aromatic group and amide region of capsaicin in the collagen-capsaicin interaction. The results may have significant implications for capsaicin-based therapeutics that target excess collagen accumulation-linked pathology, for example thrombosis, fibrosis, and sclerosis.

명태 껍질 유래 콜라겐의 분자량에 따른 이화학적 특성 및 생리활성

This study was conducted to investigate the physicochemical properties and biological activities of colla- gens with different molecular weights from Alaska pollack (Theragra chalcogramma) skin as well as their efficacies as functional materials. The molecular weights of collagens were between 1~10 kDa (below 1 kDa (AP1), 1~3 kDa (AP2), 3~10 kDa (AP3), and above 10 kDa (AP4). The protein content of AP4 (40.19 g/100 g) was the highest. Collagen contents of AP1, AP2, AP3, and AP4 were 36.43, 32.23, 19.23, and 14.89%, respectively. The free amino acid and essential amino acid contents of AP1 were higher than those of AP2, AP3, and AP4. Fourier transform infrared spectroscopy spectra of collagens with different molecular weights showed wavenumbers representing the regions of amide Ⅰ, amide Ⅱ, amide Ⅲ, and amide A, respectively. The electron-donating ability (29.51%) and SOD-like activity (38.45%) of AP1 were higher than those of AP2, AP3, and AP4. Tyrosinase inhibition activity of AP1 improved with higher treatment concentration. The rate of inhibition of MMP-1 production in HS68 cells exposed to UVB was suppressed by treatment with AP1 (29.78%) and AP2 (26.49%) at 1 mg/mL. Furthermore, there was a strong correlation between DPPH, superoxide dismutase, tyrosinase activity, and MMP-1 inhibition rate of colla- gens with different molecular weights.

Effect of KCl substitution on bacterial viability of Escherichia coli (ATCC 25922) and selected probiotics

Excessive intake of NaCl has been associated with the increased risk of several diseases, particularly hypertension. Strategies to reduce sodium intake include substitution of NaCl with other salts, such as KCl. In this study, the effects of NaCl reduction and its substitution with KCl on cell membranes of a cheese starter bacterium (Lactococcus lactis ssp. lactis), probiotic bacteria (Bifidobacterium longum, Lactobacillus acidophilus, and Lactobacillus casei), and a pathogenic bacterium (Escherichia coli) were investigated using Fourier-transform infrared (FTIR) spectroscopy. A critical NaCl concentration that inhibited the viability of E. coli without affecting the viability of probiotic bacteria significantly was determined. To find the critical NaCl concentration, de Man, Rogosa, and Sharpe (MRS) broth was supplemented with a range of NaCl concentrations [0 (control), 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, and 5.0%], and the effect on cell viability and FTIR spectra was monitored for all bacteria. A NaCl concentration of 2.5% was found to be the critical level of NaCl to inhibit E. coli without significantly affecting the viability of most of the probiotic bacteria and the cheese starter bacterium. The FTIR spectral analysis also highlighted the changes that occurred mainly in the amide regions upon increasing the NaCl concentration from 2.5 to 3.0% in most of the bacteria. Escherichia coli and B. longum were more sensitive to substitution of NaCl with KCl, compared with Lb. acidophilus, Lb. casei, and Lc. lactis ssp. lactis. To evaluate the effect of substitution of NaCl with KCl, substitution was carried out at the critical total salt concentration (2.5%, wt/vol) at varying concentrations (0, 25, 50, 75, and 100% KCl). The findings suggest that 50% substitution of NaCl with KCl, at 2.5% total salt, could inhibit E. coli without affecting the probiotic bacteria.

Low concentration structural dynamics of lanreotide and somatostatin-14.

Lanreotide, a synthetic cyclic octapeptide, analogue of the peptide hormone somatostatin-14 (SST-14), is routinely used as a long-acting medication in the management of neuroendocrine tumors. Despite its therapeutic importance, low concentration structural data is still lacking for lanreotide. In fact, the major part of the previous structural investigations were focused on the remarkable aggregation properties of this peptide, appearing at high concentrations (>5 mM). Here, we have applied three optical spectroscopic techniques, i.e. fluorescence, circular dichroism and Raman scattering, for analyzing the structural dynamics at the concentrations below 5 mM, where lanreotide exists either in a monomer state or at the first stages of aggregation. The obtained data from lanreotide were discussed through their comparison with those collected from SST-14, leading us to the following conclusions: (i) The central D-Trp residue, forming with its adjacent Lys the main receptor interacting part of lanreotide, keeps a constant high rotational freedom whatever the environment (water, water/methanol, methanol). (ii) A solvent-dependent tight β-turn, belonging to the type-II' family, is revealed in lanreotide. (iii) Raman data analyzed by band decomposition in the amide (I and III) regions allowed estimation of different secondary structural elements within the millimolar range. Interestingly, the applied protocol shows a perfect agreement between the structural features provided by the amide I and amide III Raman markers.

Characteristics and chemical composition of skins gelatin from cobia (Rachycentron canadum)

Gelatin was obtained from cobia (Rachycentron canadum) skins, which is an important commercial species for marine fish aquaculture, and it was compared with gelatin from croaker (Micropogonias furnieri) skins, using the same extraction methodology (alkaline/acid pre-treatments). Cobia skins gelatin showed values of protein yield, gelatin yield, gel strength, melting point, gelling point and viscosity higher than the values found from croaker skins gelatin. The values of turbidity and Hue angle for cobia and croaker gelatins were 403 and 74 NTU, and 84.8° and 87.3°, respectively. Spectra in the infrared region had the major absorption band in the amide region for both gelatins, but it showed some differences in the spectra. The proline and hydroxyproline contents from cobia skins gelatin (205 residues/1000 residues) was higher than from croaker skins gelatin (188 residues/1000 residues). SDS-PAGE of both gelatins showed a similar molecular weight distribution to that of standard collagen type I. Therefore, cobia skins could be used as a potential marine source of gelatin obtainment for application in diversified industrial fields.

Isolation and Characterization of Collagen from Chicken Feet

Collagen was isolated from chicken feet by using papain and pepsin enzymes in acetic acid solution at 4°C for 24h with a yield of 18.16% and 22.94% by dry weight, respectively. Chemical composition and characteristics of chicken feet collagen such as amino acid composition, SDS-PAGE patterns, FTIR spectra and thermal properties were evaluated. The chicken feet collagen is rich in the amino acids glycine, glutamic acid, proline and hydroxyproline. Electrophoresis pattern demonstrated two distinct α-chains (α1 and α2) and β chain, indicating that type I collagen is a major component of chicken feet collagen. The thermal stability of collagen isolated by papain and pepsin revealed stable denaturation temperatures of 48.40 and 53.35°C, respectively. The FTIR spectra of both collagens were similar with amide regions in A, B, I, II and III. The study demonstrated that chicken feet collagen using papain isolation method is possible as commercial alternative ingredient.

Analysis of a curve fitting model in the amide region applied to the muscle tissues of an edible fish: Labeo rohita fingerlings

Analysis of a curve fitting model in the amide region applied to the muscle tissues of an edible fish: Labeo rohita fingerlings

Production of a defensin-like antifungal protein NFAP from Neosartorya fischeri in Pichia pastoris and its antifungal activity against filamentous fungal isolates from human infections

Neosartorya fischeri NRRL 181 isolate secretes a defensin-like antifungal protein (NFAP) which has a remarkable antifungal effect against ascomycetous filamentous fungi. This protein is a promising antifungal agent of biotechnological value; however in spite of the available knowledge of the nature of its 5′-upstream transcriptional regulation elements, the bulk production of NFAP has not been resolved yet. In this study we carried out its heterologous expression in the yeast Pichia pastoris and investigated the growth inhibition effect exerted by the heterologous NFAP (hNFAP) on filamentous fungal isolates from human infections compared with what was caused by the native NFAP. P. pastoris KM71H transformant strain harboring the pPICZαA plasmid with the mature NFAP encoding gene produced the protein. The final yield of the hNFAP was sixfold compared to the NFAP produced by N. fischeri NRRL 181. Based on the signal dispersion of the amide region, it was proven that the hNFAP exists in folded state. The purified hNFAP effectively inhibited the growth of fungal isolates belonging to the Aspergillus and to the Fusarium genus, but all investigated zygomycetous strain proved to be insusceptible. There was no significant difference between the growth inhibition effect exerted by the native and the heterologous NFAP. These data indicated that P. pastoris KM71H can produce the NFAP in an antifungally active folded state. Our results provide a base for further research, e.g., investigation the connection between the protein structure and the antifungal activity using site directed mutagenesis.

Isolation and characterization of acid and pepsin-solubilized collagen from the skin of sailfish (Istiophorus platypterus)

Acid-solubilized collagen (ASC) and pepsin-solubilized collagen (PSC) were extracted from the skin of sail fish (Istiophorus platypterus) with yields of 5.76% and 2.11% respectively, on the basis of wet weight. According to the electrophoretic pattern, ASC and PSC consisted of two different α chains (α1 and α2) and were characterized as Type I collagen. Peptide maps of ASC and PSC hydrolysed by Achromopeptidase from Achromobacter lyticus exhibited similar banding patterns with human-placenta collagen suggesting, sailfish collagen is a Type I collagen. Fourier transform infrared (FTIR) spectra of ASC and PSC were quite similar and the regions of amides A, I, II and III were 3422 and 3337, 1654 and 1646, 1560 and 1559 and 1240cm−1 respectively. FTIR results suggest that the pepsin hydrolysis did not affect the secondary structure of collagen, especially triple-helical structure. 1H NMR analysis revealed the presence of water molecules along with their corresponding singlet medium chemical shift of about 4.6 to 4.8ppm in ASC and PSC. Scanning electron microscopy (SEM) studies confirmed the presence of collagen in the isolated, as fine globular filaments. Thus, possibility of sail fish collagen as an alternative source for mammalian collagen and also it could be used in nutraceutical and pharmaceutical industries.

Identification and discrimination of bacteria using Fourier transform infrared spectroscopy

Bacterial spectra were obtained in the wavenumber range of 4000–600cm−1 using FTIR spectroscopy. FTIR spectral patterns were analyzed and matched with 16S-rRNA signatures of bacterial strains OS1 and OS2, isolated from oil sludge. Specific spectral bands obtained from OS1 (FJ226761), reference strain Bacillus flexus (ATCC 49095), OS2 (FJ215874) and reference strain Stenotrophomonas maltophilia (ATCC 19861) respectively, suggested that OS1 and ATCC 49095 were closely related whereas OS2 was different. The bands probably represent groups of proteins and lipids of specific bacteria. Separate peaks found in B. flexus were similar to those of OS1. The S. maltophilia (ATCC 19861) and OS2 exhibited a similar peak at 3272cm−1. Amide bands (I, II and III) exhibited that OS1 and B. flexus were closely related, but were different from OS2. In the fingerprint region, peak at 1096cm−1 and 1360cm−1 exhibited the specific fingerprints of OS2 and reference strain S. maltophilia (ATCC 19861), respectively. The specific fingerprint signature was found at 1339cm−1 for OS1 and at 1382cm−1 for B. flexus ATCC 49095, allowing these two strains of B. flexus to be differentiated. This spectral signature originated from phospholipid and RNA components of the cell. Principle components analysis (PCA) of spectral regions exhibited with distinct sample clusters between Bacillus flexus (ATCC 49095), S. maltophilia (ATCC 19861), OS1 and OS2 in amide and fingerprint region.

Using the FT-IR linear dichroism method for molecular order determination of tendon collagen bundles and nylon 6

Polarized Fourier transform-infrared (FT-IR) was used to compare the orientation of vibrational chemical groups of bovine tendon collagen bundles (CBs) to that of nylon 6, a simpler polyamide model, in terms of linear dichroism (LD). Subtraction of spectral profiles identified the most significant differences regarding the amide regions. At 1630cm−1, the CBs displayed higher peak areas and absorbance when positioned perpendicularly (A⊥) to the plane of polarized light, in comparison with nylon 6. In contrast, at the 1526cm−1 amide II spectral region the inverse occurred. In the amide III region (1232cm−1), the LD was positive and higher for CBs. Dichroic ratios (DR=A||/A⊥) calculated from the average of ten measured spectra for CBs and nylon 6 revealed that the values for CBs were <1.0 in the 3298–1655cm−1 wavenumber range and >1.0 in the 1536–1234cm−1 wavenumber range. From 1535 to 1120cm−1, nylon 6 displayed DR values higher than those of CBs. The LD band integrated areas were higher in CBs than in nylon 6. The LD differences between CBs and nylon 6 are probably due to a more complex chemical composition and supramolecular oriented architecture in CBs in comparison to nylon 6.

Comparative study on keratin structural changes in onychomycosis and normal human finger nail specimens by Raman spectroscopy

For the purpose of investigating the secondary structural changes of keratins in onychomycosis patient nail, Raman spectra of nail clippings from onychomycosis patients were obtained and used to describe the molecular structure differences from normal nail specimens. Raman spectroscopy revealed that nail from onychomycosis patients had a significant decrease in the content of the sulfur-containing amino acids in comparison to normal nail. And the spectral analysis also demonstrated that the disulfide bonds had the trend to the energetically less favored gauche–gauche–trans (g–g–t) forms in onychomycosis patient nail samples. These results showed that the increased amounts of less stable disulfide conformers were contributed to or associated with the nail brittleness observed in onychomycosis patients. We also found that the positions of the S–S band (500–550cm−1) in onychomycosis patients were different, which gave us an indication that the disulfide groups might be related to the histopathological level and morphological characteristics. In the region of amide I, the α-helical (1652cm−1) and β-sheet (1671cm−1) content changed, these ratios (peak area of 1652cm−1 divided by peak area of the 1671cm−1) were between 0.67 and 0.75, while drastically altered ratios for the two conformers were changed to 0.25–0.63 in onychomycosis patient nail, the α-helix form seriously decreased in comparison to the normal nail. This result suggested unfolding of proteins and a more amorphous structure in the onychomycosis nail. On the other hand, the amide III (unordered) band intensity, assigned to the random coil form in normal nail clippings, at 1250cm−1 is much higher than that for the amide III (α) content (1272cm−1), and the peak area ratios of random coil band and α-helix band were about 10.00, while in the onychomycosis nail, these ratios were close to 2.00, the random coil form remarkably decreased. All the results of the present work implied that fungal erosion led to the secondary structure changes of proteins and Raman spectroscopy can be further used as a tool biomarker to diagnose this widespread disease.

Protein Dynamics of Cytochrome C Oxidase: Analysis of Vibrational Components by Phase Sensitive Detection Applied to Modulated Excitation-Seiras

Cytochrome c oxidase (CcO) from R. sphaeroides was investigated by Modulated Excitation Surface-Enhanced IR-Absorption Spectroscopy (ME-SEIRAS). Sequential electron transfer (ET) within CcO was initiated by electrochemical excitation. During the modulated excitation by periodic potential pulses with frequencies between 20 Hz and 500 Hz, time-resolved IR spectra were measured by the Step-Scan technique with time-resolution in the millisecond time range. Conformational changes of the protein structure as a result of ET lead to rather complex SEIRA spectra with many overlapping bands embedded in a broad background signal. Phase sensitive detection (PSD) was used to separate single components within the broad band of overlapping structural bands in the amide region. PSD is able to extract the periodic response of single components with the same frequency as the excitation from noise or from static background and therefore enhances the signal-to-noise ratio. Moreover, PSD enables the validation of the fit model utilized for the deconvolution of overlapping bands by analyzing the phase lags of single components acquired at different stimulation frequencies. The phase lags between the evaluated vibrational components and the modulated excitation increase with increasing excitation frequencies, which is an inherent prerequisite of the evaluation method.

Thermal, infrared spectroscopy and molecular modeling characterization of bone: An insight in the apatite-collagen type I interaction

An insight into the interaction of collagen type I with apatite in bone tissue was performed by using differential scanning calorimetry, Fourier transform infrared spectroscopy, and molecular modeling. Scanning electron microscopy shows that bone organic content incinerate gradually through the different temperatures studied. We suggest that the amide regions of the type I collagen molecule (mainly C=O groups of the peptide bonds) will be important in the control of the interactions with the apatite from bone. The amide I infrared bands of the collagen type I change when interacting to apatite, what might confirm our assumption. Bone tissue results in a loss of thermal stability compared to the collagen studied apart, as a consequence of the degradation and further combustion of the collagen in contact with the apatite microcrystals in bone. The thermal behavior of bone is very distinctive. Its main typical combustion temperature is at 360°C with a shoulder at 550°C compared to the thermal behavior of collagen, with the mean combustion peak at ca. 500°C. Our studies with molecular mechanics (MM+ force field) showed different interaction energies of the collagen-like molecule and different models of the apatite crystal planes. We used models of the apatite (100) and (001) planes; additional two planes (001) were explored with phosphate-rich and calcium-rich faces; an energetic preference was found in the latter case. We preliminary conclude that the peptide bond of collagen type I is modified when the molecule interacts with the apatite, producing a decrease in the main peak from ca. 500°C in collagen, up to 350°C in bone. The combustion might be related to collagen type I, as the ΔH energies present only small variations between mineralized and non-mineralized samples. The data obtained here give a molecular perspective into the structural properties of bone and the change in collagen properties caused by the interaction with the apatite. Our study can be useful to understand the biological synthesis of minerals as well as the organic-inorganic interaction and the synthesis of apatite implant materials.

Sub-cellular spectrochemical imaging of isolated human corneal cells employing synchrotron radiation-based Fourier-transform infrared microspectroscopy

Understanding stem cell (SC) biology remains challenging and one of the few human tissues within which their in situ location is well characterized is the cornea. Individual human corneal epithelial cells were isolated from biopsies of live tissues using fluorescence-activated cell sorting (FACS); these were divided into putative SCs, transit-amplifying (TA) cells and terminally-differentiated (TD) cells. Employing synchrotron radiation-based Fourier-transform infrared (SR-FTIR) microspectroscopy with a focal plane array (FPA), sub-cellular spatial resolution analysis of unstained isolated cells was achieved as a consequence of the brilliance of a 12 collimated beams arrangement allowing rapid spectral acquisition. Infrared (IR) spectra were extracted and pre-processed. Subsequent categorization with multivariate analysis of IR spectra derived from FPA images was used to investigate biomolecular changes between classes. A progressive segregation in cell-specific spectral categories with differentiation from SC to TA cell to TD cell was noted. Multiple different absorption peaks that discriminated putative SCs, TA cells and TD cells across DNA, protein and lipid spectral regions were identified. DNA regions (1080 and 1225 cm(-1)) and some protein regions (1443 cm(-1)) primarily segregated SCs from TA cells and TD cells, whilst amide regions and lipids (1,550, 1650 and 1740 cm(-1)) segregated TA cells and TD cells. Scanning electron microscopy images verified the external phenotypic characteristics of the different isolated cell types. These findings highlight the applicability of SR-FTIR microspectroscopy towards distinguishing SCs, TA cells and TD cells, and suggest that cellular classification via traditional methods of immunolabelling can be greatly aided by the use of spectral biomarkers.

오징어 및 명태껍질 유래 콜라겐의 추출 및 물리화학적 특성

The collagens from squid (Todarodes pacificus ) skin and Alaska pollack ( Theragra chalcogramma) skin were extracted and their physicochemical properties were investigated. The yield was improved with the increase of NaOH concentration and was increased Neutrase than Alcalase in enzymatic hydrolysates. Protein and collagen contents from Alaska pollack skin were 38.3 ∼62.7% and 13.1∼28.9%, respectively. All enzymatic hydrolysates also showed high antioxidant activities as NaOH concentration decrease. Composition of their amino acids was mainly glycine and proline. The spectrum of FT-IR of the collagen showed wavenumber at 1,631 cm-1 , 1,549 cm -1 , 1,234 cm -1 and 3,322 cm -1 representing the regions of amide Ⅰ, amide Ⅱ, amide Ⅲ and amide A, respectively. The decomposition temperature for the collagen was in the range of 300℃ and showed relatively good enough for their thermal stabilities.

Resolving Nitrogen-15 and Proton Chemical Shifts for Mobile Segments of Elastin with Two-dimensional NMR Spectroscopy

In this study, one- and two-dimensional NMR experiments are applied to uniformly (15)N-enriched synthetic elastin, a recombinant human tropoelastin that has been cross-linked to form an elastic hydrogel. Hydrated elastin is characterized by large segments that undergo "liquid-like" motions that limit the efficiency of cross-polarization. The refocused insensitive nuclei enhanced by polarization transfer experiment is used to target these extensive, mobile regions of this protein. Numerous peaks are detected in the backbone amide region of the protein, and their chemical shifts indicate the completely unstructured, "random coil" model for elastin is unlikely. Instead, more evidence is gathered that supports a characteristic ensemble of conformations in this rubber-like protein.

Green Synthesis of Silver Nanoparticles by Using Rhizome Extract of Dioscorea oppositifolia L. and their anti microbial activity against Human pathogens.

In the present investigation, synthesis of silver nanoparticles by using rhizome extracts of Dioscorea oppositifolia L. and characterized by using UV spec, FT-IR, DLS and TEM. The extract incubated with AgNo3 showed colour changed of the extract from greenish to reddish brown with intensity increasing during the period of incubation. Hence, the rhizome extract act as a reducing and capping agent. Nanoparticles were characterized by using UV visible absorption spectra has shown the absorbance peak at 409 nm, FT-IR. The synthesized silver nanoparticles were generally found to be effective as antimicrobial agents against some important human pathogens which are prevailing as antibiotic resistance. FT-IR peaks were in the extract ranging from 1000-4000 cm which confirmed the presence of polyphenols with aromatic ring and bound amide region required for the synthesis and stabilization of sliver nanoparticles.DLS shows stability of nanoparticles where as Transmission Electron Microscope confirms the sizewas about 14 nm and shape of the nanoparticle was found to be spherical in shape.

Probing the specificity of gamma-glutamylamine cyclotransferase: an enzyme involved in the metabolism of transglutaminase-catalyzed protein crosslinks

γ-Glutamylamine cyclotransferase (gGACT) catalyzes the intramolecular cyclization of a variety of L-γ-glutamylamines producing 5-oxo-L-proline and free amines. Its substrate specificity implicates it in the downstream metabolism of transglutaminase products, and is distinct from that of γ-glutamyl cyclotransferase which acts on L-γ-glutamyl amino acids. To elucidate the mechanism by which gGACT distinguishes between L-γ-glutamylamine and amino acid substrates, the specificity of the rabbit kidney enzyme for the amide region of substrates was probed through the kinetic analysis of a series of L-γ-glutamylamines. The isodipeptide N(ε)-(L-γ-glutamyl)-L-lysine 1 was used as a reference. The kinetic constants of the L-γ-glutamyl derivative of n-butylamine 7, were nearly identical to those of 1. Introduction of a methyl or carboxylate group on the carbon adjacent to the side-chain amide nitrogen in L-γ-glutamylamine substrates resulted in a dramatic decrease in substrate properties for gGACT thus providing an explanation of why gGACT does not act on L-γ-glutamyl amino acids except for L-γ-glutamylglycine. Placement of substituents on carbons further removed from the side-chain amide nitrogen in L-γ-glutamylamines restored activity for gGACT, and L-γ-glutamylneohexylamine 19 had a higher specificity constant (k(cat) /K(m)) than 1. gGACT did not exhibit any stereospecificity in the amide region of L-γ-glutamylamine substrates. In addition, analogues (26-30) with heteroatom substitutions for the γ methylene position of the L-γ-glutamyl moiety were examined. Several thiocarbamoyl derivatives of L-cysteine (28-30) were excellent substrates for gGACT.