Purification and Characterization of 5,10-Methenyltetrahydrofolate Synthetase from Chicken Liver

조용권,Cho, Yong-Kweon Korean Chemical Society 2010 대한화학회지 Vol.54 No.5

5,10-Methenyltetrahydrofolate synthetase from chicken liver was purified through 30-70% ammonium sulfate fractionation, Q Sepharose Fast Flow anion exchange and Source 15Phe hydrophobic interaction chromatography. Specific activities of cell extract, ammonium sulfate, Q Sepharose Fast Flow and Source 15Phe were 0.0085, 0.031, 0.80 and 1.27 U/mg, respectively. Purification fold activities of cell extract, ammonium sulfate, Q Sepharose Fast Flow and Source 15Phe were 1, 3.7, 94.1 and 149.4, respectively. HPLC gel permeation chromatography and SDS-polyacrylamide electrophoresis experiments indicated that the enzyme is a monomeric protein with a molecular weight of 22.8 kDa. Km for 5-methyl THF and Mg-ATP were $7.1\;{\mu}M$ and $63\;{\mu}M$, respectively. Optimum temperature and pH were $30^{\circ}C$ and 6.0, respectively. The data for metal ion specificity and stoichiometry showed that the maximum activity was obtained with a 1:l. ratio of $Mg^{2+}$. The ATP and Km values increased in the order of MgATP, MgCTP, MgUTP and MgGTP, and the maximum activities also decreased in the same order, indicating MgATP as the most efficient substrate. The enzyme was chemically modified only by tetranitrometane and 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide, indicating that tyrosine and carboxylate are present in the active site. 닭의 간으로부터 5,10-methenyltetrahydrofolate synthetase를 30-70% 황산암모늄 분획, Q Sepharose Fast Flow anion exchange and Source 15Phe hydrophobic interaction chromatography을 이용하여 정제하였다. 세포 추출물, 황산암모늄 분획, Q Sepharose Fast Flow와 Source 15Phe 단계에서의 비활성은 각각0.0085, 0.031, 0.80 및 1.27 U/mg 이었다. 세포 추출물, 황산암모늄 분획, Q Sepharose Fast Flow와 Source 15Phe 단계에서의 정제도는 각각 1, 3.7, 94.1 및 149.4 이었다. HPLC gel permeation chromatography와 SDS-polyacrylamide electrophoresis 실험으로부터 5,10-methenyltetrahydrofolate synthetase는 분자량이 22.8 kDa인 단량체임을 알 수 있었다. 5-methyl THF과 Mg-ATP의 Km은 각각 $7.1\;{\mu}M$ 및 $63\;{\mu}M$ 이었다. 최적온도와 최적pH는 각각 $30^{\circ}C$ 및 6.0 이었다. 금속이온에 대한 특이성과 스토키오메트리 실험으로부터 최고속도가 $Mg^{2+}$과 1:1일 때 얻어진다는 것을 알 수 있었다. ATP와 Km은 MgATP, MgCTP, MgUTP 및 MgGTP의 순서로 증가하였으며 최고 역가는 같은 순으로 감소하였는데, 이는 MgATP 가 가장 효과적인 기질임을 증명한다. 이 효소는 tetranitrometane 및 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide에 의해서만 수식되었는데, 이는 tyrosine and carboxylate 잔기가 효소의 활성부위에 존재함을 나타낸다.

위에서 발생한 선-편평세포암종

조용권,안지영,홍성권,최민규,노재형,손태성,김성,Cho, Yong-Kwon,An, Ji-Yeong,Hong, Seong-Kweon,Choi, Min-Gew,Noh, Jae-Hyung,Sohn, Tae-Sung,Kim, Sung 대한위암학회 2006 대한위암학회지 Vol.6 No.1

배경: 위암은 조직학적으로 선암종이 가장 흔하며 선-편평세포암종은 전체 위암의 0.5%를 차지하는 드문 질환으로 알려져 있다. 그러나 선-편평세포암종의 발생기전 및 임상병리학적 특성에 대하여 명확히 알려진 것이 없으며 치료 방법 역시 명확히 정립되지 못하였다. 이에 본원에서 체험한 선-편평세포암종을 정리하여 보고하고자 한다. 대상 및 방법: 1994년 9월부터 2004년 12월까지 삼성서울병원 외과에서 위암으로 수술 받은 8,268명의 환자 중 병리조직학적 검사상 선-편평세포암종으로 확진된 8명의 환자를 의무기록을 바탕으로 후향적으로 조사하였다. 결과: 남자가 5명, 여자가 3명이었으며 중앙 연령은 49 ($41{\sim}69$)세였다. 종양의 위치는 중부가 3명, 하부가 5명이었고 크기는 평균 6.2 ($2.5{\sim}8cm$)였다. 병기는 UICC 분류상 II기가 5명, III기가 2명, IV기가 1명이었으며 조직 검사상 림프절 전이가 있었던 경우는 7명에서 관찰되었고 전이 림프절 개수는 평균 3.7 ($1{\sim}14$)개였다. IV기 환자 1명은 위절제술을 시행하지 못하고 위-공장우회술만 시행하였고 수술 후 5개월만에 사망하였다. 근치적 위절제술과 항암 화학요법을 시행한 6명의 환자들의 중앙 생존 기간은 34 ($12{\sim}66$)개월이었으며 1명은 경과 관찰 중 손실되었고 2명은 수술 후 각각 30개월과 34개월에 재발로 사망하였으며 나머지 4명은 재발의 증거 없이 외래 추적 관찰 중이다. 결론: 위에서 발생하는 선-편평세포암종도 선암종과 마찬가지로 림프절 절제를 포함한 근치적 위절제술과 함께 술 후 보조 항암 화학 요법 등의 적극적인 치료가 필요할 것으로 생각된다.X>$1,000{\mu}g$을 1개월 간격으로 $5{\sim}6$회 근육 주사하면 정상 혈중 농도를 유지할 수 있다.사료된다. 실험동물모델의 신경연접 변화를 분석하는 것은 신경연접의 형태적 가소성을 이해하는데 이바지할 것으로 생각된다.용하게 활용될 수 있는 자료가 될 것이다. 척도(r=-.341, p=0.036)는 보호자의 나이가 많을수록 점수가 낮았다. 5) 사회적 관계 영역(영역 3)과 관련 있는 요인은 없었으나, 하부척도 중 성적활동 척도는 교육년수가 길수록 높은 점수를 보이고 있었다(r=0.344, p=0.037). 6) 환경 영역(영역 4)은 교육년수가 길수록 점수가 높았지만(r=0.482, p=0.003), 환아의 나이가 많을수록 낮은 삶의 질 수준을 보고하였다(r=0.328, p=0.044). 한편 하부 척도 중에서는 신체적 안전 척도(r=-0.414, p=0.010), 거주환경 척도(r=-0.429, p=0.007), 새로운 정보나 기술의 취득 척도(r=-0.382, p=0.018), 의료서비스 및 사회보장서비스 척도(r=-0.351, p=0.031)가 환아의 나이와 음의 상관관계를 보였으며, 신체적 안전척도는 보호자의 나이가 많을수록 삶의 질이 낮음을 보고하였다(r=-403, p=0.012). 한편, 새로운 정보나 기술의 취득척도(r=0.406, p=0.013), 여가활동 척도(r=0.464, p=0.004), 교통 척도(r=0.363, p=0.027)은 교육연수가 길수록 높은 점수를 보고하였다. 결론: 주의력결핍 과잉행동장애 환아의 보호자가 느끼는 주관적인 삶의 질은 건강대조군에 비해 나쁘지 않았다. 그러나 환아의 나이가 많을수록, 보호자의 교육연수가 낮을수록 스스로 느끼는 삶의 질의정도가 낮았으므로 이에 대한 관심이 필요할 Purpose: A primary adenosquamous carcinoma of the stomach is relatively rare, accounting for only about 0.5% of all gastric cancers. However, its histopathologic characteristics are still unclear, and the most appropriate form of therapy has not been established yet. Materials and Methods: We retrospectively reviewed the clinicopathologic features of 8 patients with pathologically confirmed primary adenosquamous carcinomas out of 8,268 patients who underwent gastric cancer surgery at Samsung Medical Center between September 1994 and December 2004. Results: The median age of the 8 patients was 49 ($41{\sim}69$) years, and the male : female ratio was 5 : 3. In 3 patients, the tumor was located at the mid body of the stomach, and in 5 patients, at the lower body or antrum. The tumor sizes were $2.5{\sim}8cm$. Seven patients showed metastases to the regional lymph nodes. The UICC stage distribution were: 5 stage II, 2 stage III, and 1 stage IV. In the stage IV patient, a palliative gastrojejunostomy was performed, and he died 5 months after surgery. Of the 7 patients who underwent a radical gastrectomy and adjuvant chemotheratpy, the median survival was 34 ($12{\sim}66$) months, 2 patients died of cancer recurrence, and 4 patients are being followed up without evidence of recurrence. Conclusion: As for an adenocarcinoma of the stomach, a radical gastrectomy including regional lymph node dissection and postoperative adjuvant therapy should be performed for appropriate treatment of an adenosquamous carcinoma of the stomach.

반하에서 3,4-Dihydroxybenzaldehyde의 분리, 구조동정 및 Tyrosinase 활성 저해효과

조용권 ( Yong Kweon Cho ),신동수 ( Dong Soo Shin ),백도현 ( Do Hyeon Paik ),윤도영 ( Do Young Yoon ),신동민 ( Dong Min Shin ) 대한본초학회 2006 大韓本草學會誌 Vol.21 No.2

Kinetic and Chemical Mechanism of Pig Liver Methenyltetrahydrofolate Synthetase

Yong Kweon Cho(조용권) 한국생명과학회 2008 생명과학회지 Vol.18 No.8

30~70% 황산암모늄 분획, Fast Q 음이온 교환수지 및 Phenyl Agarose 수지를 통해 돼지간유래의 methenyltetrahydrofolate synthetase를 정제하였으며 정제도는 119이었다. SDS-PAGE 및 HPLC를 이용한 gel permeation column chromatography에 의해 이 효소는 분자량이 23 kDa인 단량체로 확인되었다. 최적 온도와 최적 pH는 각각 35℃ 와 6.5이었다. 이 효소는 tetranitromethane 및 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide(EDC)에 의해서만 활성이 감소되었는데, 이는 티로신과 카르복실산이 효소의 활성부위에 존재한다는 것을 나타낸다. 활성부위의 pH 실험으로부터 2개의 티로신이 기질의 결합에 관여하며 하나의 카르복실기가 촉매반응에 관여한다는 사실이 관찰되었다. 따라서 이 효소는 2개의 티로신이 ATP와 5-formylTHF과 결합하며 하나의 카르복실기가 일반염기로서 촉매한다는 것을 알 수 있다. Methenyltetrahydrofolate synthetase extract was obtained from mouse liver and purified via 30~70% ammonium sulfate fractionation, Fast Q anion exchange and phenyl agarose chromatography. HPLC gel chromatography and SDS-polyacrylamide electrophoresis experiments showed that the enzyme is a monomer with molecular weight of 23 kDa. Optimum temperature and pH were 35℃ and 6.5, respectively. The enzyme was chemically modified only by tetranitromethane and 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide (EDC), indicating that tyrosine and carboxylate are in the active site. pH studies showed that 2 tyrosines are involved in the binding of the substrates and a carboxylate in catalysis. Therefore, the chemical mechanism of the enzyme is likely that 2 tyrosines bind to ATP and 5-formylTHFand a carboxylate acts as a general base.

Propionibacterium fruedenreichii 에서 분리한 무기인산 의존성 Phophofructokinase 의 금속이온 요구성

조용권 ( Yong Kweon Cho ) 생화학분자생물학회 1992 BMB Reports Vol.25 No.2

Exchange inert Cr(H₂O)PPi was synthesized to study metal requirement of inorganic pyrophosphate-dependent phosphofructokinase from Propionibacterium freudenreichii. Cr(H₂O)₄PPi acted as a competitive inhibitor with two inhibition constants (K_(i,1)=25μM, K_(i,2)=115 μM), indicating the possibility of the presence of the second binding site for pyrophosphate. To test the possibility of the second metal ion requirement, Cr(H₂O)₄PPi was added to the reaction mixture after the addition of MgC1₂ and vice versa. There was no activity with the spectroscopic method. To use the more sensitive method, [^(32)P]Cr(H₂O)₄PPi was synthesized and incubated in the presence of MgC1₂. It was found that Cr(H₂O)₄PPi acted as a substrate but with only a thousandth of regular enzyme activity in the presence of the second metal ion, indicating that PPi-PFK from P. freudenreichii requires at least two metal ions.

박하에서 분리한 Caffeic Acid Methyl Ester가 자궁경부암 바이러스 발암단백질 E6의 기능에 미치는 영향

백태웅,이경애,안민정,주혜경,오현철,안종석,조용권,명평근,윤도영,Baek, Tae-Woong,Lee , Kyung-Ae,Ahn , Min-Jung,Joo, Hae-Hyung,Oh , Hyun-Cheol,Ahn , Jong-Seok,Cho , Yong-Kweon,Myung , Pyung-Keun,Yoon, Do-Young 대한약학회 2004 약학회지 Vol.48 No.6

Cervical cancer is one of the leading causes of female death. Viral oncoproteins E6 and E7 are selectively retained and expressed in carcinoma cells infected with HPV (Human pa pilloma virus) type 16 and cooperated in immotalization and transformation of primary keratinocyte. E6 and E7 oncoproteins interfere the functions of tumor suppressor proteins p53 and retinoblasoma protein (pRb), respectively. Among a lots of natural products, Mentha arvensis Linne var.piperascens have inhibitory effects on bindings between E6 oncoprotein and tumor suppressor p53, E3 ubiqutin- protein ligase (E6AP). HPV oncoprotein inhibitors from Mentha piperita L. were isolated by solvent partition and column chromatography (Silica gel, RP-18) and inhibitory compounds were finally purified by HPLC using an ELISA screening system based on binding between E6 and E6AP. The aim of this study is to identify the structure of inhibitory compounds and to investigate whether these compounds have inhibitory effects on functions of E6 oncoprotein. We investigated whether caffeic acid methyl ester (CAM) extracted from Mentha piperita L. could inhibit the function of E6 oncoprotein. CAM inhibited the in vitro binding of E6 and E6AP which are essential for the binding and degradation of the tumor suppressor p53 and also inhibited the proliferation of human cervical cancer cell lines (SiHa and CaSKi) in a dose response manner. These results suggest that CAM inhibited the function of E6 oncoprotein, suggesting that it can be used as a potential drug for the treatment of cervical cancers infected with HPV.

쇠별꽃 메탄올추출물의 항산화능 및 B16F10 세포에서 광보호 효능

전근영 ( Geun Yeong Jeon ),진방민 ( Bang Min Jin ),조용권 ( Yong Kweon Cho ),김영철 ( Young Chul Kim ) 대한미용학회(구 대한미용과학회) 2021 대한미용학회지 Vol.17 No.2

The aim of the current study was to investigate the antioxidant ability of Stellaria aquatica L. methanol extract (SAME) and its photoprotection efficacy of B16F10 cells irradiated with UVA. Total polyphenol and flavonoid contents in SAME were estimated to be 284.4 TAE mg/g and 210.5 RE mg/g, respectively. At 1,000 μg/mL, the electron-donating ability of SAME was found to be 58.0% and its SC50 for free radical scavenging was 748.2 μg/ mL. The viabilities of B16F10 cells treated with SAME at 25 and 50 μg/mL were 87.7% and 73.1%, respectively. On the other hand, the viabilities of B16F10 cells treated with IBMX at 25 and 50 μg/mL were 81.3% and 76.1%, respectively. The maximum permissible levels for treating the B16F10 cells with SAME and IBMX were calculated to be 25 μg/mL. Irradiation with 5 and 10 mJ/㎠ UVA decreased the viabilities of the B16F10 cells by 21.6% and 25.1%, respectively, which, however, increased viabilities by 13.0% (p < 0.001) and 11.5% (p < 0.001), respectively on treatment with 25 μg/mL SAME. On the other hand, when 5 and 10 mJ/㎠ UVA-irradiated B16F10 cells was treated with 25 μg/mL IBMX, the viabilities increased by 8.7% (p < 0.05) and 4.7% (p < 0.01), respectively. These results suggest that SAME can be used as a natural cosmetic ingredient for photoprotection of melanocytes from UVA irradiation.

계피로부터 Malassezia 종에 대한 항균물질 분리 및 동정

엄지영(Ji-Young Um),진방민(Bang-Min Jin),조용권(Yong-Kweon Cho) 한국화장품미용학회 2020 한국화장품미용학회지 Vol.10 No.3

To overcome side effects of dandruff medications, many researches on natural anti-dandruff compounds are actively being conducted. Here, we tried to isolate and identify antifungal compounds against four Malassezia species (M. restricta, M. globosa, M. furfur, M. sympodialis) from Cinnamomoum cassia Blume. Cinnamomum cassia Blume was extracted with 80% (v/v) ethanol for 7 days. The extracts were purified by solvent fractionation, silica gel column chromatography and HPLC. The purified compound was identified for the active peak with 1H-NMR and <SUP>13</SUP>C-NMR. Among 5 solvent fractions, only hexane fraction (CHF) showed antifungal activity. The data showed that CHF has higher activity than that of Zinc pyrithione (ZPT) in M. furfur, and lower than that of ZPT in M. restricta, M. globosa and M. sympodialis. Thus, CHF has been further purified by silica gel column chromatography and Prep-HPLC. The analysis of 1H-NMR and <SUP>13</SUP>C-NMR revealed that structure of isolated compound is coumarin. Coumarin showed the antifungal activity of 11.5 mm, 10.1 mm, 14.2 mm and 19.8 mm for M. furfur, M. restricta, M. globosa and M. sympodialis, respectively. MIC50 values of coumarin were 79.09 μg/mL, 133.91 μg/mL, 63.41 μg/mL and 19.72 μg/mL for M. furfur, M. restricta, M. globosa and M. sympodialis, respectively. The anti-inflammatory activity of coumarin at concentrations of 0.013∼0.855 nM showed that coumarin inhibited nitric oxide production by 18∼22% in RAW 264.7 cell. These data suggest that Cinnamomum cassia Blume extracts and its isolated coumarin are good candidates for dandruff medications.

The Signal Sequence of Sporulation-Specific Glucoamylase Directs the Secretion of Bacterial Endo-1,4-β-D-Glucanase in Yeast

Soon-Cheol Ahn(안순철),Eun-Ju Kim(김은주),Sung-Sik Chun(전성식),Yong-Kweon Cho(조용권),Ja-Young Moon(문자영),Dae-Ook Kang(강대욱) 한국생명과학회 2012 생명과학회지 Vol.22 No.2

효모 Saccharomyces diastaticus가 포자형성기에 세포질에서 생산된다고 알려진 포자형성 특이 glucoamylase (SGA)가 세포 외로 분비되는 단백질임을 증명하고자 S. dastaticus의 SGA promoter와 예상되는 분비신호서열 다음에 reporter gene으로 사용한 고초균의 CMCase 구조유전자를 융합한 재조합 플라스미드 pYSC25를 제작하고 수주세포인 S. diastaticus YIY345에 형질전환 하였다. 형질전환체를 1% CMC를 포함하는 최소한천배지에서 배양한 후 Congo red 염료로 염색하여 생성된 투명환으로부터 SGA의 분비서열에 의해 세균의 CMCase가 효모세포외로 분비되는 것을 확인하였다. 효모세포부위 별 CMCase의 활성분포를 측정하여 SGA 분비서열의 분비효율을 추정하기 위해 효모세포 배양액을 배양상등액, periplasmic 및 세포질 분획으로 나눈 다음 효소활성을 측정한 결과 CMCase 활성의 76%가 배양상등액과 periplasmic 부위에 존재하였으며 N-연결형 당쇄가 일어났으므로 SGA분비서열은 효과적으로 작용함을 알 수 있었다. 대조균인 고초균에서 생산된 CMCase에서는 당쇄가 일어나지 않은 것을 확인하였다. 이상의 결과로부터 SGA는 아미노 말단에 존재하는, 24개의 아미노산으로 구성된 분비서열을 보유한 분비성 단백질임을 확인하였다. The sporulation-specific glucoamylase (SGA) of Saccharomyces diastaticus is known to be produced in the cytoplasm during sporulation. For the purpose of proving that SGA has secretory potential, we constructed a hybrid plasmid, pYESC25, containing the promoter and the putative signal sequence of the SGA fused in frame to the endo-1,4- -D-glucanase (CMCase) gene of Bacillus subtilis without its own signal sequence. The recipient yeast strain of S. diastaticus YIY345 was transformed with the hybrid plasmid. CMCase secretion from S. diastaticus harboring pYESC25 into culture medium was confirmed by the formation of yellowish halos around transformants after staining with Congo red on a CMC agar plate. The transformant culture was fractionated to the extracellular, periplasmic, and intracellular fraction, followed by the measurement of CMCase activity. About 63% and 13% enzyme activity were detected in the culture supernatant (extracellular fraction) and periplasmic fraction, respectively. Furthermore, ConA-Sepharose chromatography, native gel electrophoresis, and activity staining revealed that CMCase produced in yeast was glycosylated and its molecular weight was larger than that of the unglycosylated form from B. subtilis. Taking these findings together, SGA has the potential of secretion to culture medium, and the putative signal sequence of SGA can efficiently direct bacterial CMCase to the yeast secretion pathway.

Inhibition and Chemical Mechanism of Protocatechuate 3,4-dioxygenase from Pseudomonas pseudoalcaligenes KF707

Taekyeong Kang(강태경),Sang Ho Kim(김상호),Mi Ja Jung(정미자),Yong Kweon Cho(조용권) 한국생명과학회 2015 생명과학회지 Vol.25 No.5

Pseudomonas pseudoalcaligenes KF707에서 정제한 protocatechuate 3,4-dioxygenase의 특징을 조사하기 위하여 pH안정성, 화학적 저해, 화학적 수식과 pH의존성 반응 상수에 대한 실험을 수행하였다. 이 효소는 pH 4.5~10.7에서 안정하였다. L-ascorbate와 glutathione은 Kis가 각각 0.17 mM과 0.86 mM인 경쟁적 저해제였으며, DL-dithiothreitol은 Kis 1.57 mM 및 Kii 8.08 mM의 비경쟁적 저해패턴을 나타내었다. Potassium cyanide, p-hydroxybenzoate 및 sodium azide는 Kis가 각각 55.7 mM, 0.22 mM 및15.64 mM이었으며, Kii는 각각94.1 mM, 8.08 mM, 및 662.64 mM인 비경쟁적 저해패턴을 나타내었다. FeCl2는 Kis가 29 μM로 가장 우수한 경쟁적 저해제였으며, FeCl3, MnCl2, CoCl2, HgCl2, AlCl3도 각각 Kis가 1.21 mM, 0.85 mM, 3.98 mM, 0.17 mM 및 0.21 mM인 경쟁적 저해패턴을 보였다. 한편, 다른 금속이온들은 비경쟁적 저해패턴을 나타내었다. pH의존성 반응상수의 실험결과로부터 pK 6.2와 9.4의 촉매부위와 pK 5.5와 9.0의 결합부위가 존재함을 알 수 있었다. Lysine, cysteine, tyrosine, carboxyl과 histidine은 각각의 고유한 화학적 수식제에 의해 수식되었는데, 이는 이들 잔기들이 결합과 촉매에 관여한다는 것을 나타낸다. 위 결과를 토대로 화학적 메커니즘을 제시한다. We carried out pH stability, chemical inhibition, chemical modification, and pH-dependent kinetic parameter assessments to further characterize protocatechuate 3,4-dioxygenase from Pseudomonas pseudoalcaligenes KF707. Protocatechuate 3,4-dioxygenase was stable in the pH range of 4.5~10.5. L-ascorbate and glutathione were competitive inhibitors with Kis values of 0.17 mM and 0.86 mM, respectively. DL-dithiothreitol was a noncompetitive inhibitor with a Kis value of 1.57 mM and a Kii value of 8.08 mM. Potassium cyanide, p-hydroxybenzoate, and sodium azide showed a noncompetitive inhibition pattern with Kis values of 55.7 mM, 0.22 mM, and 15.64 mM, and Kii values of 94.1 mM, 8.08 mM, and 662.64 mM, respectively. FeCl2 was the best competitive inhibitor with a Kis value of 29 μM. FeCl3, MnCl2, CoCl2, and AlCl3 were also competitive inhibitors with Kis values of 1.21 mM, 0.85 mM, 3.98 mM, and 0.21 mM, respectively. Other metal ions showed noncompetitive inhibition patterns. The pH-dependent kinetic parameter data showed that there may be at least two catalytic groups with pK values of 6.2 and 9.4 and two binding groups with pK values of 5.5 and 9.0. Lysine, cysteine, tyrosine, carboxyl, and histidine were modified by their own specific chemical modifiers, indicating that they are involved in substrate binding and catalysis.