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Mechanistic consequences of replacing the active-site nucleophile Glu-358 in Agrobacterium sp. beta-glucosidase with a cysteine residue.

ABSTRACT: Retaining glycosidases achieve the hydrolysis of glycosidic bonds through the assistance of two key active-site carboxyls. One carboxyl functions as a nucleophile/leaving group, and the other acts as the acid-base catalyst. It has been suggested that a cysteine residue could fulfil the role of the active site nucleophile [Hardy and Poteete (1991) Biochemistry 30, 9457-9463]. To test the validity of this proposal, a kinetic evaluation was conducted on the active-site nucleophile cysteine mutant (Glu-358-->Cys) of the retaining beta-glucosidase from Agrobacterium sp. The Glu-358-->Cys mutant was able to complete the first step (glycosylation) of the enzymic mechanism, forming a covalent glycosyl-enzyme intermediate, but the rate constant for this step was decreased to 1/10(6) of that of the native enzyme. The subsequent hydrolysis (deglycosylation) step was also severely affected by the replacement of Glu-358 with a cysteine residue, with the rate constant being depressed to 1/10(7) or less. Thus Cys-358 functions inefficiently in both the capacity of catalytic nucleophile and leaving group. On the basis of these results it seems unlikely that the role of the active-site nucleophile in retaining glycosidases could successfully be filled by a cysteine residue.

SUBMITTER: Lawson SL

PROVIDER: S-EPMC1219128 | biostudies-other | 1998 Feb

REPOSITORIES: biostudies-other

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Project description:The aim of this study was to investigate the effect of replacing Glu in the Lys-urea-Glu PSMA-targeting pharmacophore of [68Ga]Ga-HTK03041 with a close analog on the uptake of kidneys, salivary glands and PSMA-expressing tumor xenografts. Methods: HTK03161, HTK03149 and HTK03189A/B were obtained by replacing Glu in HTK03041 with Asp, Aad (L-2-aminoadipic acid) and Api (2-aminopimelic acid), respectively. PSMA binding affinities were measured by competition binding assays. PET imaging and biodistribution studies of 68Ga-labeled ligands were performed in LNCaP tumor-bearing mice. The best candidate HTK03149 was selected and radiolabeled with 177Lu, and SPECT imaging and biodistribution studies were performed in LNCaP tumor-bearing mice. Radiation dosimetry calculation was conducted using the OLINDA software. Radioligand therapy study was performed in LNCaP tumor-bearing mice treated with [177Lu]Lu-HTK03149 (9.3-148 MBq), [177Lu]Lu-PSMA-617 (37 MBq) or natLu-HTK03149 (500 pmol). Results: PSMA binding affinities (Ki) of Ga-HTK03161, Ga-HTK03149, Ga-HTK03189A and Lu-HTK03149 were 3.88±0.66, 6.99±0.80, 550±35.7 and 1.57±0.42 nM, respectively. PET imaging showed that all 68Ga-labeled HTK03161, HTK03149 and HTK03189A/B were excreted mainly via the renal pathway and had minimal uptake in all organs/tissues including kidneys and salivary glands. Tumor xenografts were clearly visualized in PET images of [68Ga]Ga-HTK03161 and [68Ga]Ga-HTK03149 but were barely visualized using [68Ga]Ga-HTK03189A/B. Tumor uptake values for [68Ga]Ga-HTK03161, [68Ga]Ga-HTK03149, [68Ga]Ga-HTK0189A and [68Ga]Ga-HTK03189B were 12.7±1.91, 19.1±6.37, 2.10±0.28 and 0.67±0.15 %IA/g, respectively at 1h post-injection, and their average kidney and salivary gland uptake values were 2.13-4.41 and 0.20-0.23 %IA/g, respectively. Longitudinal SPECT imaging studies showed that [177Lu]Lu-HTK03149 was excreted mainly through the renal pathway with high uptake in LNCaP tumors and minimal uptake in all normal organs/tissues. The tumor uptake of [177Lu]Lu-HTK03149 peaked at 4h post-injection (20.9±2.99 %IA/g) and the uptake was sustained over time. Compared to [177Lu]Lu-PSMA-617, [177Lu]Lu-HTK03149 had 145% increase in tumor absorbed dose but 70% less in kidney absorbed dose, leading to an 7.1-fold increase in tumor-to-kidney absorbed dose ratio. Radioligand therapy studies showed that only half of the [177Lu]Lu-PSMA-617 injected dosage was needed for [177Lu]Lu-HTK03149 to achieve the same median survival. Conclusion: Replacing Glu in the PSMA-targeting Lys-urea-Glu pharmacophore of [68Ga]Ga-HTK03041 with Asp and Aad generates [68Ga]Ga-HTK03161 and [68Ga]Ga-HTK03149, respectively, and the new derivatives retain high uptake in LNCaP tumors and have minimal uptake in normal organs/tissues including kidneys and salivary glands. [177Lu]Lu-HTK03149 also retain high uptake in LNCaP tumors and has minimal uptake in normal organs/tissues, and is, therefore, promising for clinical translation to treat prostate cancer.

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Mechanistic consequences of replacing the active-site nucleophile Glu-358 in Agrobacterium sp. beta-glucosidase with a cysteine residue.

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